Methods for enhancing genome stability and telomere elongation in embryonic stem cells

ABSTRACT

The disclosure provides methods for increasing genome stability of an embryonic stem (ES) cell or induced pluripotent stem (iPS) cell, increasing telomere length in an ES or iPS cell, or both, for example by contacting an ES or iPS cell with an agent that increases expression of Zscan4 in the cell. Methods for increasing the genome stability in a population of ES or iPS cells, increasing telomere length in a population of ES or iPS cells, or both, are provided, for example by selecting Zscan4 +  ES or iPS cells from the population of ES or iPS cells (which can include both Zscan4 +  and Zscan4 −  ES or iPS cells). Therapeutic methods of using ES or iPS cells expressing Zscan4 are also provided. Further provided are methods of treating cancer by administering a Zscan4 polynucleotide or Zscan4 polypeptide. Also provided are methods of inducing differentiation of isolated ES or iPS cells into germ cells.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is the U.S. National Stage of International ApplicationNo. PCT/US2010/047644, filed Sep. 2, 2010, which was published inEnglish under PCT Article 21(2), which claims the benefit of U.S.Provisional Application No. 61/275,983, filed Sep. 4, 2009, which isherein incorporated by reference in its entirety.

FIELD

This disclosure relates to embryonic stem (ES) cells and inducedpluripotent stem (iPS) cells and the role of Zscan4 expression inpromoting genome stability and telomere elongation in ES and iPS cells.

BACKGROUND

Mouse embryonic stem (ES) cells are derived from the inner cell mass(ICM) of blastocysts and share similar gene expression patterns with theICM. The defining features of ES cells are pluripotency andself-renewal, both of which have been the focus of intensive researchfor many years. Another hallmark of mouse ES cells is their ability todefy cellular senescence and to proliferate more than 250 doublingswithout crisis or transformation (Suda et al., J Cell Physiol133:197-201, 1987). Although the fraction of euploid cells tends todecrease in long-term culture (Rebuzzini et al., Cytotechnology58:17-23, 2008), the genome integrity of mouse ES cells is more strictlymaintained than any other cultured cells. For example, ES cells maintaintheir ability to form chimeric animals with germline competency evenafter many passages (Longo et al., Transgenic Res 6:321-328, 1997; Nagyet al., Proc Natl Acad Sci USA 90:8424-8428, 1993; Sugawara et al., CompMed 56:31-34, 2006). The mutation frequency in ES cells is also muchlower (>100-fold) than those in mouse embryonic fibroblast cells andother somatic cells (Cervantes et al., Proc Natl Acad Sci USA99:3586-3590, 2002). The unique feature of mouse ES cells can be furtherhighlighted by a lower frequency of chromosomal abnormalities comparedto embryonal carcinoma cells, which share similar characteristics to EScells (Blelloch et al., Proc Natl Acad Sci USA 101:13985-13990, 2004),as well as some human ES cells (Brimble et al., Stem Cells Dev13:585-597, 2004). However, the mechanism by which mouse ES cellsmaintain genomic stability is currently poorly understood.

Telomeres are repetitive DNA sequences accompanied by proteins that capand protect the end of each chromosome from continuous degradation ineach cell cycle, thereby securing and protecting chromosomal integrity.Telomere shortening may lead to cancer by contributing to genomicinstability (Raynaud et al., Crit. Rev Oncol Hematol 66:99-117, 2008),and has been associated with aging and cellular senescence (Yang,Cytogenet Genome Res 122:211-218, 2008). Telomerase has been identifiedas the major enzyme known to be involved in telomere elongationmaintenance. Although telomerase is active in ES cells (Thomson et al.,Science 282:1145-1147, 1998), telomerase knockout ES cells (Terc^(−/−))show a marked reduction in telomere length only after 400 celldoublings, reaching a dramatic senescence event 10-30 doublings later,followed by establishment of a telomerase-independent population with nomarked short telomeres (Niida et al., Nat Genet. 19:203-206, 1998; Niidaet al., Mol Cell Biol 20:4115-4127, 2000). Hence, atelomerase-independent mechanism for telomere maintenance, namedalternative lengthening of telomeres (ALT) (Bryan et al., EMBO J.14:4240-4248, 1995), has been suggested for Terc^(−/−) ES cells.Apparently, telomere recombination, or telomere sister chromatidexchange (T-SCE) can compensate for the lack of telomerase activity.Indeed, an increased frequency of T-SCE events has been demonstrated inlong-term cultures of Terc^(−/−) ES cells (Bailey et al., Nucleic AcidsRes 32:3743-3751, 2004; Wang et al., Proc Natl Acad Sci USA102:10256-10260, 2005).

Additionally, telomere recombination appears to be a normal mechanism inpreimplantation embryos. Even though enzymatic activity of telomerasecannot be detected in preimplantation embryos from the unfertilizedoocyte stage to the blastocyst stage (Wright et al., Dev Genet.18:173-179, 1996), telomere length is rapidly increased during thisperiod. In just one cell cycle, the average telomere length of 2-cellstage mouse embryos is doubled compared to that of unfertilized eggs(Liu et al., Nat Cell Biol 9:1436-1441, 2007). Although T-SCE eventshave been demonstrated in several studies, genes involved in thisimportant process remain to be identified.

SUMMARY

The present disclosure provides methods for increasing genome stabilityof an embryonic stem (ES) cell or induced pluripotent stem (iPS) cell,or increasing telomere length in an ES or iPS cell, or both. Forexample, such methods can enhance the presence of a normal karyotype,reduce chromosome fusions and fragmentations, reduce genomic sisterchromosome exchange, increase telomere recombination, or combinationsthereof, in a cell. In particular examples, methods include contactingan ES or iPS cell with an agent (for example introducing the agent intoan ES or iPS cells) that increases expression of Zscan4 in the ES or iPScell relative to expression of Zscan4 in a cell in the absence of theagent. Exemplary agents that increase Zscan4 expression include but arenot limited to Zscan4-encoding nucleic acid molecules, retinoic acidsand agents that induce oxidative stress.

Methods for increasing the genome stability in a population of ES or iPScells, increasing telomere length in a population of ES or iPS cells, orboth, are provided. In particular examples, methods include selectingZscan4⁺ ES or iPS cells from the population of ES or iPS cells (whichmay include both Zscan4⁺ and Zscan4⁻ ES or iPS cells). For example, apopulation of ES or iPS cells can be transfected with an expressionvector that includes a Zscan4 promoter operably linked to a reportergene, wherein expression of the reporter gene indicates Zscan4 isexpressed in the subpopulation of ES or iPS cells. Cells expressing thereporter gene can be detected (e.g., by detecting a signal produced bythe protein encoded by the reporter gene) and then isolated. Forexample, if the reporter gene is green fluorescent protein (GFP) or arelated fluorescent protein (such as Emerald), Zscan4-positive cells canbe recognized based on the fluorescence and can be sorted by afluorescence-activated cell sorter (FACS). If the reporter gene is acell surface marker, Zscan4-positive cells can be sorted by FACS or bymagnetic beads that can bind to the cell surface marker.

ES or iPS cells expressing Zscan4 can be used therapeutically. Forexample, a subject in need of ES cell therapy can be selected andadministered a therapeutically effective amount of a subpopulation ofundifferentiated ES or iPS cells that are Zscan4⁺. For example, cellsfully differentiated from Zscan4⁺ ES or iPS cells can also be used fortherapeutic administration. Examples of subjects that can benefit fromsuch therapy include a subject having cancer, an autoimmune disease, aneurologic injury or a neurodegenerative disorder, as well as otherdisorders that can benefit from regenerative therapies.

Also provided herein is a method of treating a subject with cancer byadministering to the subject an agent that increases expression ofZscan4.

Further provided is a method of inducing differentiation of isolated EScells or isolated iPS cells into germ cells. In some embodiments, themethod includes contacting the ES or iPS cells with an agent thatincreases expression of Zscan4 in the ES or iPS cells, thereby inducingdifferentiation of the ES or iPS cells into germ cells.

A method of inducing meiosis, meiosis-specific recombination and/or DNArepair in an isolated ES cell or an isolated iPS cell is also provided.In some embodiments, the method includes contacting the ES or iPS cellwith an agent that increases expression of Zscan4 in the ES or iPS cell,thereby inducing meiosis, meiosis-specific recombination and/or DNArepair in the ES or iPS cell.

A method of protecting a cell from a DNA-damaging agent by contactingthe cell with an agent that increases expression of Zscan4 is alsoprovided.

The foregoing and other objects and features of the disclosure willbecome more apparent from the following detailed description, whichproceeds with reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A is a pair of digital images showing expression of Zscan4 ishighly heterogeneous in mouse ES cell colonies, as demonstrated by RNAwhole-mount in situ hybridization (WISH), whereas that of control Pou5f1is homogeneous. FIG. 1B is a schematic diagram of the Zscan4-promoterEmerald-reporter vector. FIG. 1C is a pair of digital images showingvisualization of Emerald expression under the Zscan4 promoter inpZscan4-Emerald cells by phase-contrast fluorescent microscope (leftpanel) and confocal microscope section (right panel). All nuclei werelabeled with DAPI. FIG. 1D is a FACS plot of pZscan4 Emerald cells. FIG.1E shows FACS-sorting of pZscan4-Emerald cells into four groupsaccording to their fluorescent intensities (upper panel). The expressionlevels of Zscan4c in each cell population were measured by qRT-PCR(lower panel). FIGS. 1F-1H are a series of FACS plots showing atime-course FACS analysis of pZscan4-Emerald cells. FIG. 1I is a scatterplot showing a pair-wise comparison of gene expression profiles betweenEmerald(+) and Emerald(−) cells by DNA microarray analysis. Color-codedspots are genes that are differentially expressed with statisticalsignificance (FDR<0.05 and fold-change >1). FIG. 1J is a table of thetop 20 differentially expressed genes in Em⁺ cells.

FIG. 2A is a series of digital images showing RNA in situ hybridizationof Zscan4 and 8 additional genes (Tcstv1, Eif1a, Pif1, AF067063,EG668777, LOC332923, BC061212, and EG627488), selected for upregulationin Em(+) cells and showing a “Zscan4-like” expression pattern. Controlgenes are Krt2-8/EndoA, a trophectoderm and visceral-endoderm markerthat stains differentiated cells surrounding undifferentiated EScolonies; and Pou5f1 (Oct4 or Oct3/4), as a pluripotency marker. FIG. 2Bis a set of digital images of double-fluorescence RNA in situhybridization showing co-expression of Zscan4 transcript (FITC) andtranscripts of: Tcstv3, Eifla, AF067063, EG668777, LOC332923, BC061212and EG627488. FIG. 2C is a series of bar graphs showing qRT-PCR analysisof Zscan4 and six other selected genes upregulated in Em(+) cells(EG627488, Arg2, AF067063, Gm428, Tcstyl/3, BC061212). Results fromunfertilized oocytes (MII), 1-cell embryos (1), early 2-cell embryos(E2), late 2-cell embryos (L2), 4-cell embryos (4), 8-cell embryos (8),Morulae (M) and Blastocysts (BL) are shown.

FIG. 3A is a schematic diagram of the pZscan4-CreERT2 vector. Thecre-recombinase gene is expressed under the Zscan4 promoter. In thepresence of tamoxifen, CreERT2 fusion enzyme translocates into thenucleus, excises the neomycin resistance gene upstream of LacZ gene, andactivates LacZ expression. FIG. 3B is a set of digital images ofRNA-FISH for Zscan4 and immunostaining for Cre-recombinase, which showsco-staining of Zscan4 RNA and Cre-recombinase in a small population ofcells after short exposure of pZscan4-CreERT2 ES cells to tamoxifen.FIG. 3C is a set of digital images showing co-immunostaining analysis ofPou5f1 and LacZ after long-term exposure of pZscan4-CreERT2 ES cells totamoxifen. All nuclei were labeled with DAPI. FIG. 3D is a series ofphase contrast images of pZscan4-CreERT2 cells maintained in thepresence of tamoxifen for up to 9 passages (27 days) and subsequentlystained with X-gal to demonstrate LacZ activity. By passage 9, themajority of the cells are marked by LacZ. FIG. 3E is a series of FACSplots showing analysis of LacZ expression by CMFDG-staining. Acontinuous increase in the LacZ-positive cell population in thecontinuous presence of tamoxifen was observed. FIG. 3F is a graphshowing LacZ-positive cells counted by two different methods: FACSanalysis using CMFDG and manual counting after staining cells withX-gal. Both methods show similar results with linear increase ofLacZ-positive cells with passages. FIG. 3G is a series of digital imagesshowing pZscan4-CreERT2 LacZ-positive cells are able to contribute toall three major cell lineages by embryoid body (EB) differentiationassay. Phase contrast images of X-gal staining of the cells showLacZ-positive Zscan4-daughter cells in endoderm, ectoderm (epithelia,neural rosettes by the 3rd day of differentiation, neurons by the 7thday of differentiation) and mesoderm (beating muscles). FIG. 3H is aseries of digital images showing co-immunostaining for LacZ anddifferent germ layer markers. Zscan4-daughter cells (marked by LacZexpression) are able to contribute to all three germ layers: Dab2 forendoderm; ASM-1 for mesoderm; and Nestin for neuroectoderm. FIG. 3I isan image of a gel showing genotyping of 9 embryos (E10.5), whichdemonstrates chimerism in 8 out of the 9 embryos tested.

FIG. 4A is a pair of graphs showing qRT-PCR analysis demonstratingZscan4 shRNA vector downregulated Zscan4 expression by ˜90%, whereasZscan4c-ORF induction by Dox removal rescued Zscan4 expression (upperpanel). shRNA against Luciferase was used in the same parental cells asnegative control. qRT-PCR analysis showed Zscan4c over-expression led toZscan4d induction (lower panel); both paralogs were knocked down byshRNA. FIG. 4B is a pair of representative images of Zscan4-knockdowncells and the cells rescued by Zscan4-ORF induction by the removal ofDox for 3 days. Venus was used as a reporter for gene induction. FIG. 4Cis a schematic illustration of Zscan4 knockdown phenotype. Cellspresented normal colony morphology during the first few passages;however, 8 passages after clone isolation (approximately 31 celldoublings), cell culture crisis was observed. The surviving cells couldbe maintained, but their doubling time was abnormally long. FIG. 4D is agraph showing reduced cell proliferation by Zscan4 knockdown. For eachpassage, 4×10⁵ cells were plated and cells were counted after 3 days inculture. FIG. 4E is a graph showing reduced proliferation by Zscan4knockdown at passage 7, prior to cell culture crisis (∘, Δ), whereasrescue of Zscan4 improved proliferation rate (•, ▴). Controls includedTet-Empty cells in Dox+ (⋄) and Dox− (♦); tet-Zscan4c cells in Dox+ (Δ)and Dox− (▴); and shCont cells in Dox+ (□) and Dox− (▪). Assays wereperformed in biological triplicate in two independent experiments. FIG.4F is a graph showing increased apoptosis in Zscan4-knockdown cells.Apoptosis assay by Annexin-V was performed by FACS analysis. Controlsincluded tet-Zscan4c cells (Zscan4) for basal apoptosis levels; shRNAcontrol cells (shCont) for possible off-target effects; and tet-Emptycells (Empty) for doxycycline effect. Apoptotic cells were visualized byV-PE-conjugated Annexin-V antibody as well as the cell impermeant dye(7-AAD) as an indicator for dead cells. Dead cells were excluded to givethe total number of apoptotic-live cells in culture. FIG. 4G is a tableshowing karyotype analysis of Zscan4 knockdown and rescue cells comparedto shRNA controls. Results from passage 3 (left panel) show multiplekaryotype abnormalities, such as fusions and fragmentations, which arepartially prevented by Zscan4 rescue. Results from passage 10 after cellcrisis (right panel) show further deterioration of karyotype (only 30%normal), which are partially prevented by Zscan4 rescue.

FIG. 5A is a series of images showing karyotype instability ofZscan4-knockdown cells. Shown are representative telomere FISH images inmetaphase chromosome spreads stained by DAPI. Left two panels are imagesof Zscan4-knockdown cells. White arrows indicate missing or very shorttelomeres. Fusion indicates fused chromosomes. Right two panels areshRNA control cells with normal telomeres and Zscan4 rescue cells withimproved telomere length and karyotype. FIG. 5B is a graph showingrelative telomere length ratio (T/S) measured by qPCR analysis ofZscan4-knockdown cells before (passage 6) and after (passage 9) of cellculture crisis. Relative telomere length ratio (T/S) was calculated bynormalizing telomere length by a single copy gene. Error bars indicateS.E.M. FIG. 5C is a pair of graphs showing Q-FISH, performed onZscan4-knockdown cells, which confirmed a significant telomereshortening. A distribution diagram of relative telomere length is theresult of analyzing 10 pooled nuclei (total of 1600 telomeres) by Q-FISHand TFL-Telo software. X-axis: Telomere fluorescence unit (1 TFU≈1 kb).FIG. 5D is a pair of representative images of telomere FISH inZscan4c-overexpressing cells: Cy3-conugated PNA-telomere probes andDAPI. FIG. 5E is a pair of graphs showing a distribution of relativetelomere length in Zscan4c-overexpressing cells. FIG. 5F is a graphshowing relative telomere length ratio (T/S) measured by qPCR analysisof Zscan4c-overexpressing cells and FACS-sorted pZscan4-Emerald cells(Em+ and Em−). Error bars indicate S.E.M.

FIG. 6A is a graph showing telomerase activity measured by TRAP assay.FIG. 6B is a series of representative images of tet-Zscan4c cells inDox+ and Dox− conditions: ZSCAN4C-FLAG is visualized byAlexa546-conugated antibody; Venus reporter; and all nuclei visualizedby DAPI. FIG. 6C is a set of images showing co-localization ofZSCAN4C-FLAG in sister chromatids in metaphase spreads. Chromosomes arestained by DAPI. Arrows mark chromosomes with intense staining at thetelomere regions. FIG. 6D is a set of images showing increased incidenceof T-SCE in Zscan4-overexpressing cells. Representative images oftelomere recombination are visualized by chromosome orientationFISH(CO-FISH) assay. Chromosomes are stained by DAPI. A Cy3-cojugatedtelomere probe marks telomeres. Left panel: tet-Zscan4c cells (Dox+)with no telomere recombination in most nuclei. Middle and Right panels:test-Zscan4c cells (Dox−) with increased telomere recombination events(arrowheads). Images are representative of three independentexperiments. FIG. 6E is a table showing a more than 10-fold increase inT-SCE frequency by Zscan4 over-expression in tet-Zscan4c cells. Thetable shows the total T-SCE events observed in >20 nuclei per sample. Asnegative controls, tet-Zscan4c cells in the Dox+ condition and tet-Emptycells for a possible doxycycline effect on T-SCE, were used.

FIG. 7A is a set of images showing the increased rate of sisterchromatid exchange (SCE) in Zscan4-knockdown cells. Representativeimages of SCE assay performed in Zscan4-knockdown cells are shown.Arrows mark SCE events. FIG. 7B is a graph showing SCE assay results,which demonstrate a 2.5-fold increase in the percentage of cellspresenting SCE and genomic instability in Zscan4 knockdown cells. TheSCE experiment was done in 3 independent experiments with the analysisof 50 metaphases for each experiment (n=50, total n=150). Error barsindicate S.E.M. FIG. 7C is a graph showing that the number of SCE eventsper affected metaphases was also significantly elevated. Error barsindicate S.E.M. FIG. 7D is a schematic diagram showing the feature ofES* state and the effect of Zscan4 knockdown on ES cells.

FIG. 8A is a series of images showing immunostaining and co-localizationof ZSCAN4 with the telomere markers TRF1 (upper panel) and TRF2 (lowerpanel), as demonstrated by confocal microscopy. Nuclei are indicated byDAPI. Size bar=10 μm. FIG. 8B is a graph showing the results of qPCRanalysis, which confirmed Zscan4 induced the upregulation ofmeiosis-specific homologous recombination genes Spo11, Dmc 1 and Smc1β.FIG. 8C is a series of images showing immunostaining analysis byconfocal microscopy. ZSCAN4 foci co-localize with SPO11 (upper panel)and in most cells with γ-H2AX foci (lower panel). FIG. 8D is a series ofimages showing telomere localization of ZSCAN4 with TRF1 (upper panel)and DMC1/γ-H2AX foci (lower panel) in pZscan4-Emerald cells.

FIG. 9 is a line graph showing the percentage of Emerald⁺ cellsfollowing treatment of MC1-ZE7 cells in the presence or absence ofleukemia inhibitory factor (LIF) and in the presence or absence ofretinoic acid (RA). Cells were passaged to gelatin-coated plates in thepresence of LIF (LIF+) and absence of atRA (atRA-). The next day (Day0), the culture medium of each well was changed to four differentconditions: LIF+atRA−, LIF+atRA+, LIF− atRA−, and LIF− atRA+. The cellswere maintained in the same culture medium for 8 days with the mediumchanges every day, but without passaging. Cells were harvested every dayand the number of Emerald GFP⁺ cells was measured by flow cytometry.

FIG. 10A is a line graph showing the effect of different retinoids(atRA, 9-cis RA, 13-cis RA or vitamin A) on the percentage of Zscanecells in culture. Shown is the percentage of Emerald⁺ MC1-ZE-7 cells upto seven days following exposure to the retinoid. FIG. 10B is a linegraph showing the effect of the different reintoids on ES cellproliferation.

FIG. 11 is a series of flow cytometry plots showing induction of Zscan4expression in response to oxidative stress. MC1-ZE7 cells were culturedin standard ES cell medium (LIF+). Hydrogen peroxide (H₂O₂) was added tothe medium at a final concentration of 100 μM, 300 μM, or 1000 μM. Thecells were cultured for two days and the fraction of Emerald⁺ (i.e.,Zscane cells) was measured by flow cytometry.

FIGS. 12A and 12B are graphs showing survival of control ES cells (A)and tet-Zscan4 ES cells (B) after treatment with mitomycin C (MMC).Control ES cells contained a control plasmid (tet-Empty). Cells werecultured in standard ES medium (LIF+) and passaged into two groups: (1)in the absence of doxycycline (Dox) or (2) in the presence of Dox at afinal concentration of 0.2 μg/ml. The Dox+ and Dox− media were changedevery day. On the fourth day, the cells were cultured for 6 hours in thepresence of MMC at a final concentration ranging from 0 to 600 ng/ml.MMC was removed from the culture by changing the media and cells werethen incubated for 3 more days in the Dox+ medium, with the mediumchanged every day. Cells were harvested and the number of live cells wascounted.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand.

The Sequence Listing is submitted as an ASCII text file, Feb. 24, 2012,88.4 KB, which is incorporated by reference herein.

In the accompanying sequence listing:

SEQ ID NOs: 1 and 2 are the nucleotide sequences of the forward andreverse primers, respectively, used for amplification of the Zscan4promoter.

SEQ ID NOs: 3-20 are the nucleotide sequences of qPCR primers.

SEQ ID NOs: 21 and 22 are primer sequences used to amplify Zscan4shRNAs.

SEQ ID NO: 23 is the sequence of a nucleotide repeat used for CO-FISHanalysis.

SEQ ID NOs: 24 and 25 are nucleotide and amino acid sequences ofZscan4a.

SEQ ID NOs: 26 and 27 are nucleotide and amino acid sequences ofZscan4b.

SEQ ID NOs: 28 and 29 are nucleotide and amino acid sequences ofZscan4c.

SEQ ID NOs: 30 and 31 are nucleotide and amino acid sequences ofZscan4d.

SEQ ID NOs: 32 and 33 are nucleotide and amino acid sequences ofZscan4e.

SEQ ID NOs: 34 and 35 are nucleotide and amino acid sequences ofZscan4f.

SEQ ID NOs: 36 and 37 are nucleotide and amino acid sequences of humanZSCAN4, deposited under Genbank Accession No. NM_(—)152677 as of Sep. 4,2009.

SEQ ID NO: 38 is the nucleotide sequence of the Zscan4-Emeraldexpression vector (9396 bp). The starting nucleotide of the Zscan4cpromoter sequence is 906 and the ending nucleotide is 4468.

SEQ ID NO: 39 is an N-terminal epitope of Zscan4 used to generateantibodies.

DETAILED DESCRIPTION I. Introduction

The Zscan4 gene was previously identified using expression profiling ofall preimplantation stages of mouse embryos using a large-scale cDNAsequencing project (Ko et al., Development 127:1737-1749, 2000; Sharovet al., PLoS Biol 1:E74, 2003; WO 2008/118957) and DNA microarrayanalysis (Hamatani et al., Dev Cell 6:117-131, 2004). Zscan4 consists of6 paralog genes (Zscan4a to Zscan4f) and 3 pseudogenes (Zscan4-ps1 toZscan4-ps3) clustered on an approximately 850 kb region of chromosome 7.Among the six paralogs, the open reading frames of Zscan4c, Zscan4d, andZscan4f encode a SCAN domain as well as all four zinc finger domains,suggesting their potential role as transcription factors. A highexpression peak of Zscan4 marks the late 2-cell stage of mouse embryos.Zscan4 expression, normally below detection threshold in blastocysts, isreactivated in vitro in a small fraction of ES cells in culture.Although all six Zscan4 paralogs are expressed in ES cells, Zscan4c isthe predominant paralog, whereas Zscan4d is the predominant paralog in2-cell embryos (Falco et al., Dev Biol 307:539-550, 2007).

Disclosed herein is the finding that Zscan4 is associated with a uniquetransient state in undifferentiated ES cells, in which other 2-cellembryo-specific genes are activated. Furthermore, Zscan4 was determinedto be essential for long-term maintenance of genomic integrity and tomediate a regulated telomere recombination in normal undifferentiated EScells, therefore making it indispensable for proper long-termself-renewal in ES cells. Also disclosed herein is the finding thatZscan4 is involved in the induction and recruitment of themeiosis-specific homologous recombination machinery to telomeres. Theinventors have further determined that Zscan4 expression can be inducedby retinoids or oxidative stress and expression of Zscan4 protects cellsagainst DNA-damaging agents.

II. Abbreviations

ALT alternative lengthening of telomeres

atRA all-trans retinoid acid

bp base pair

BrdU bromodeoxyuridine

BSA bovine serum albumin

cDNA complementary DNA

CO-FISH chromatid orientation FISH

DNA deoxyribonucleic acid

Dox doxycycline

DSB double strand DNA break

EB embryoid bodies

EC embryonal carcinoma

EG embryonic germ

Em(+) emerald-positive

Em(−) emerald-negative

ES embryonic stem

ES* zscan4⁺ ES cells

FACS fluorescence activated cell sorting

FBS fetal bovine serum

FISH fluorescence in situ hybridization

GS germline stem

GFP green fluorescent protein

hCG human chorionic gonadotropin

ICM inner cell mass

iPS cells induced pluripotent stem cells

IRES internal ribosomal entry site

IU international unit

LIF leukemia inhibitory factor

maGSC multipotent adult germline stem cell

MAPC multipotent adult progenitor cell

ORF open reading frame

PCR polymerase chain reaction

PFA paraformaldehyde

PMSG pregnant mare's serum gonadotropin

Q-FISH quantitative FISH

qPCR quantitative polymerase chain reaction

qRT-PCR quantitative reverse-transcriptase polymerase chain reaction

RA retinoic acid

RNA ribonucleic acid

SEM standard error of the mean

shRNA short hairpin RNA

SSC saline-sodium citrate

SCE sister chromatid exchange Tet tetracycline

TFU telomere fluorescence unit

TRAP telomeric repeat amplification protocol

TS trophoblast stem

T-SCE telomere sister chromatid exchange

USSC unrestricted somatic stem cell

UTR untranslated region

UV ultraviolet

WISH whole-mount in situ hybridization

II. Terms and Methods

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in molecular biology maybe found in Benjamin Lewin, Genes V, published by Oxford UniversityPress, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of the various embodiments, the followingexplanations of specific terms are provided:

Administration: To provide or give a subject an agent, such as an EScell or population of ES cells that express Zscan4, by any effectiveroute. An exemplary route of administration includes, but is not limitedto, injection (such as subcutaneous, intramuscular, intradermal,intraperitoneal, intravenous or intra-arterial).

Adult stem cell: Undifferentiated cells, found throughout the body afterembryonic development, that multiply by cell division to replenish dyingcells and regenerate damaged tissues. Also known as somatic stem cells.

Agent: Any protein, nucleic acid molecule, compound, small molecule,organic compound, inorganic compound, or other molecule of interest. Insome embodiments, the “agent” is any agent that increases expression ofZscan4. In particular example, the agent is a nucleic acid moleculeencoding Zscan4, a retinoid or an agent that induces oxidative stress.

Autoimmune disease: A disease resulting from an aberrant immuneresponse, such as the production of antibodies or cytotoxic T cellsspecific for a self antigen or a subject's own cells or tissues.

Cancer: A malignant tumor characterized by abnormal or uncontrolled cellgrowth. Other features often associated with cancer include metastasis,interference with the normal functioning of neighboring cells, releaseof cytokines or other secretory products at abnormal levels andsuppression or aggravation of inflammatory or immunological response,invasion of surrounding or distant tissues or organs, such as lymphnodes, etc. “Metastatic disease” refers to cancer cells that have leftthe original tumor site and migrate to other parts of the body forexample via the bloodstream or lymph system.

Contacting: Placement in direct physical association; includes both insolid and liquid form. As used herein, “contacting” is usedinterchangeably with “exposed.”

Degenerate variant: A polynucleotide encoding a polypeptide, such as aZscan4 polypeptide, that includes a sequence that is degenerate as aresult of the genetic code. There are 20 natural amino acids, most ofwhich are specified by more than one codon. Therefore, all degeneratenucleotide sequences are included as long as the amino acid sequence ofthe polypeptide encoded by the nucleotide sequence is unchanged.

Differentiation: Refers to the process by which a cell develops into aspecific type of cell (for example, muscle cell, skin cell etc.). In thecontext of the present disclosure, differentiation of embryonic stemcells refers to the development of the cells toward a specific celllineage. As a cell becomes more differentiated, the cell loses potency,or the ability to become multiple different cell types. As used herein,inhibiting differentiation means preventing or slowing the developmentof a cell into a specific lineage.

DNA repair: Refers to a collection of processes by which a cellidentifies and corrects damage to the DNA molecules that encode itsgenome.

Embryonal carcinoma (EC) cells: Pluripotent stem cells derived fromteratocarcinomas, which are considered the malignant counterparts ofembryonic stem (ES) cells.

Embryonic stem (ES) cells: Pluripotent cells isolated from the innercell mass of the developing blastocyst. ES cells can be derived from anyorganism, such as a mammal. In one embodiment, ES cells are producedfrom mice, rats, rabbits, guinea pigs, goats, pigs, cows, monkeys andhumans. Human and murine derived ES cells are exemplary. ES cells arepluripotent cells, meaning that they can generate all of the cellspresent in the body (bone, muscle, brain cells, etc.). Methods forproducing murine ES cells can be found, for example, in U.S. Pat. No.5,670,372. Methods for producing human ES cells can be found, forexample, in U.S. Pat. No. 6,090,622, PCT Publication No. WO 00/70021 andPCT Publication No. WO 00/27995. A number of human ES cell lines areknown in the art and are publically available. For example, the NationalInstitutes of Health (NIH) Human Embryonic Stem Cell Registry provides alist of a number of human ES cell lines that have been developed (a listcan be found online at the NIH Office of Extramural Research website athttp://grants.nih gov/stem_cells/registry/current.htm).

Encapsulated: As used herein, a molecule “encapsulated” in ananoparticle refers to a molecule (such as Zscan4 protein) that iseither contained within the nanoparticle or attached to the surface ofthe nanoparticle, or a combination thereof.

ES cell therapy: A treatment that includes administration of ES cells toa subject. In particular examples, the ES cells are Zscan4⁺, wherein theZscan4 is endogenous or exogenous to the ES cells.

Fluorescent protein: A genetically-encoded protein that exhibitsfluorescence when exposed to a particular wavelength of light. A broadrange of fluorescent protein genetic variants have been developed thatfeature fluorescence emission spectral profiles spanning almost theentire visible light spectrum. Examples include anthozoan fluorescentproteins, green fluorescent protein (GFP) (which exhibits greenfluorescence when exposed to blue light), as well as mutants thereofsuch as EGFP, blue fluorescent protein (EBFP, EBFP2, Azurite, mKalama1,which except for mKalama1 contain a Y66H substitution), cyan fluorescentprotein (ECFP, Cerulean, CyPet, which include a Y66W substitution), andyellow fluorescent protein derivatives (YFP, Citrine, Venus, YPet, whichinclude a T203Y substitution). Other particular examples include EmeraldGreen Fluorescent Protein (EmGFP) and Strawberry. For overview, see forexample Shaner et al., Nat. Methods 2(12):905-909, 2005.

Genome stability: The ability of a cell to faithfully replicate DNA andmaintain integrity of the DNA replication machinery. An ES cell with astable genome generally defies cellular senescence, can proliferate morethan 250 doublings without undergoing crisis or transformation, has alow mutation frequency and a low frequency of chromosomal abnormalities(e.g., relative to embryonal carcinoma cells), and maintains genomicintegrity. Long telomeres are thought to provide a buffer againstcellular senescence and be generally indicative of genome stability andoverall cell health. Chromosome stability (e.g., few mutations, nochromosomal rearrangements or change in number) is also associated withgenome stability. A loss of genome stability is associated with cancer,neurological disorders and premature aging. Signs of genome instabilityinclude elevated mutation rates, gross chromosomal rearrangements,alterations in chromosome number, and shortening of telomeres.

Germ cell: The cells that give rise to the gametes (i.e., eggs andsperm) of organisms that reproduce sexually.

Heterologous: A heterologous polypeptide or polynucleotide refers to apolypeptide or polynucleotide derived from a different source orspecies. For example, a mouse Zscan4 peptide expressed in a human EScell is heterologous to that ES cell.

Host cells: Cells in which a vector can be propagated and its DNAexpressed. The cell may be prokaryotic or eukaryotic. The term alsoincludes any progeny of the subject host cell. It is understood that allprogeny may not be identical to the parental cell since there may bemutations that occur during replication. However, such progeny areincluded when the term “host cell” is used.

Induced pluripotent stem (iPS) cells: A type of pluripotent stem cellartificially derived from a non-pluripotent cell, typically an adultsomatic cell, by inducing a “forced” expression of certain genes. iPScells can be derived from any organism, such as a mammal. In oneembodiment, iPS cells are produced from mice, rats, rabbits, guineapigs, goats, pigs, cows, monkeys and humans. Human and murine derivediPS cells are exemplary. In particualr examples, iPS cells are used inplace of (or in addition to) the ES cells described herein. For exampleiPS cells that are Zscane can be used in place of (or in addition to)the Zscan4⁺ ES cells.

iPS cells are similar to ES cells in many respects, such as theexpression of certain stem cell genes and proteins, chromatinmethylation patterns, doubling time, embryoid body formation, teratomaformation, viable chimera formation, and potency and differentiability.Methods for producing iPS cells are known in the art. For example, iPScells are typically derived by transfection of certain stemcell-associated genes (such as Oct-3/4 (Pouf51) and Sox2) intonon-pluripotent cells, such as adult fibroblasts. Transfection can beachieved through viral vectors, such as retroviruses, lentiviruses, oradenoviruses. For example, cells can be transfected with Oct3/4, Sox2,Klf4, and c-Myc using a retroviral system or with OCT4, SOX2, NANOG, andLIN28 using a lentiviral system. After 3-4 weeks, small numbers oftransfected cells begin to become morphologically and biochemicallysimilar to pluripotent stem cells, and are typically isolated throughmorphological selection, doubling time, or through a reporter gene andantibiotic selection. In one example, iPS from adult human cells aregenerated by the method of Yu et al. (Science 318(5854):1224, 2007) orTakahashi et al. (Cell 131(5):861-72, 2007).

Isolated: An isolated nucleic acid has been substantially separated orpurified away from other nucleic acid sequences and from the cell of theorganism in which the nucleic acid naturally occurs, i.e., otherchromosomal and extrachromosomal DNA and RNA. The term “isolated” thusencompasses nucleic acids purified by standard nucleic acid purificationmethods. The term also embraces nucleic acids prepared by recombinantexpression in a host cell as well as chemically synthesized nucleicacids. Similarly, “isolated” proteins have been substantially separatedor purified from other proteins of the cells of an organism in which theprotein naturally occurs, and encompasses proteins prepared byrecombination expression in a host cell as well as chemicallysynthesized proteins. Similarly, “isolated” cells, such as thoseexpressing Zscan4, have been substantially separated away from othercell types (such as cells that don't express Zscan4).

Meiosis: The process of reductional division in which the number ofchromosomes per cell is cut in half. In animals, meiosis always resultsin the formation of gametes, while in other organisms it can give riseto spores. As with mitosis, before meiosis begins, the DNA in theoriginal cell is replicated during S-phase of the cell cycle. Two celldivisions separate the replicated chromosomes into four haploid gametesor spores. Meiosis is essential for sexual reproduction and thereforeoccurs in all eukaryotes (including single-celled organisms) thatreproduce sexually. During meiosis, the genome of a diploid germ cell,which is composed of long segments of DNA packaged into chromosomes,undergoes DNA replication followed by two rounds of division, resultingin four haploid cells. Each of these cells contains one complete set ofchromosomes, or half of the genetic content of the original cell.

Multipotent cell: Refers to a cell that can form multiple cell lineages,but not all cell lineages.

Nanoparticle: A particle less than about 1000 nanometers (nm) indiameter. Exemplary nanoparticles for use with the methods providedherein are made of biocompatible and biodegradable polymeric materials.In some embodiments, the nanoparticles are PLGA nanoparticles. As usedherein, a “polymeric nanoparticle” is a nanoparticle made up ofrepeating subunits of a particular substance or substances. “Poly(lacticacid) nanoparticles” are nanoparticles having repeated lactic acidsubunits. Similarly, “poly(glycolic acid) nanoparticles” arenanoparticles having repeated glycolic acid subunits.

Neurologic injury: A trauma to the nervous system (such as to the brainor spinal cord or particular neurons), which adversely affects themovement and/or memory of the injured patient. For example, suchpatients may suffer from dysarthria (a motor speech disorder),hemiparesis or hemiplegia.

Neurodegenerative disorder: A condition in which cells of the brain andspinal cord are lost. Neurodegenerative diseases result fromdeterioration of neurons or their myelin sheath which over time lead todysfunction and disabilities. Conditions that result can cause problemswith movement (such as ataxia) and with memory (such as dementia).

Non-human animal: Includes all animals other than humans. A non-humananimal includes, but is not limited to, a non-human primate, a farmanimal such as swine, cattle, and poultry, a sport animal or pet such asdogs, cats, horses, hamsters, rodents, such as mice, or a zoo animalsuch as lions, tigers or bears. In one example, the non-human animal isa transgenic animal, such as a transgenic mouse, cow, sheep, or goat. Inone specific, non-limiting example, the transgenic non-human animal is amouse.

Operably linked: A first nucleic acid sequence is operably linked to asecond nucleic acid sequence when the first nucleic acid sequence isplaced in a functional relationship with the second nucleic acidsequence. For instance, a promoter is operably linked to a codingsequence if the promoter affects the transcription or expression of thecoding sequence. Generally, operably linked nucleic acid sequences arecontiguous and where necessary to join two protein coding regions, inthe same reading frame.

Oxidative stress: An imbalance between the production of reactive oxygenspecies and a biological system's ability to readily detoxify thereactive intermediates or to repair the resulting damage. Disturbancesin the normal redox state of tissues can cause toxic effects through theproduction of peroxides and free radicals that damage all components ofthe cell, including proteins, lipids, and DNA. In some embodiments ofthe disclosed methods, the agent that induces oxidative stress ishydrogen peroxide (H₂O₂).

Pharmaceutically acceptable carriers: The pharmaceutically acceptablecarriers of use are conventional. Remington's Pharmaceutical Sciences,by E. W. Martin, Mack Publishing Co., Easton, Pa., 15th Edition (1975),describes compositions and formulations suitable for pharmaceuticaldelivery of the Zscan4 proteins, Zscan4 nucleic acid molecules, or cellsherein disclosed.

In general, the nature of the carrier will depend on the particular modeof administration being employed. For instance, parenteral formulationsusually comprise injectable fluids that include pharmaceutically andphysiologically acceptable fluids such as water, physiological saline,balanced salt solutions, aqueous dextrose, glycerol or the like as avehicle. For solid compositions (e.g., powder, pill, tablet, or capsuleforms), conventional non-toxic solid carriers can include, for example,pharmaceutical grades of mannitol, lactose, starch, or magnesiumstearate. In addition to biologically-neutral carriers, pharmaceuticalcompositions to be administered can contain minor amounts of non-toxicauxiliary substances, such as wetting or emulsifying agents,preservatives, and pH buffering agents and the like, for example, sodiumacetate or sorbitan monolaurate.

Pharmaceutical agent: A chemical compound, small molecule, or othercomposition capable of inducing a desired therapeutic or prophylacticeffect when properly administered to a subject or a cell. “Incubating”includes a sufficient amount of time for a drug to interact with a cell.“Contacting” includes incubating a drug in solid or in liquid form witha cell.

Pluripotent cell: Refers to a cell that can form all of an organism'scell lineages (endoderm, mesoderm and ectoderm), including germ cells,but cannot form an entire organisms autonomously.

Polynucleotide: A nucleic acid sequence (such as a linear sequence) ofany length. Therefore, a polynucleotide includes oligonucleotides, andalso gene sequences found in chromosomes. An “oligonucleotide” is aplurality of joined nucleotides joined by native phosphodiester bonds.An oligonucleotide is a polynucleotide of between 6 and 300 nucleotidesin length. An oligonucleotide analog refers to moieties that functionsimilarly to oligonucleotides but have non-naturally occurring portions.For example, oligonucleotide analogs can contain non-naturally occurringportions, such as altered sugar moieties or inter-sugar linkages, suchas a phosphorothioate oligodeoxynucleotide. Functional analogs ofnaturally occurring polynucleotides can bind to RNA or DNA, and includepeptide nucleic acid (PNA) molecules.

Polypeptide: A polymer in which the monomers are amino acid residueswhich are joined together through amide bonds. When the amino acids arealpha-amino acids, either the L-optical isomer or the D-optical isomercan be used, the L-isomers being preferred. The terms “polypeptide” or“protein” as used herein are intended to encompass any amino acidsequence and include modified sequences such as glycoproteins. The term“polypeptide” is specifically intended to cover naturally occurringproteins, as well as those which are recombinantly or syntheticallyproduced.

The term “polypeptide fragment” refers to a portion of a polypeptidewhich exhibits at least one useful epitope. The term “functionalfragments of a polypeptide” refers to all fragments of a polypeptidethat retain an activity of the polypeptide, such as a Zscan4.Biologically functional fragments, for example, can vary in size from apolypeptide fragment as small as an epitope capable of binding anantibody molecule to a large polypeptide capable of participating in thecharacteristic induction or programming of phenotypic changes within acell, including affecting cell proliferation or differentiation. An“epitope” is a region of a polypeptide capable of binding animmunoglobulin generated in response to contact with an antigen. Thus,smaller peptides containing the biological activity of Zscan4, orconservative variants of Zscan4, are thus included as being of use.

The term “soluble” refers to a form of a polypeptide that is notinserted into a cell membrane.

The term “substantially purified polypeptide” as used herein refers to apolypeptide which is substantially free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In one embodiment, the polypeptide is at least 50%, for example at least80% free of other proteins, lipids, carbohydrates or other materialswith which it is naturally associated. In another embodiment, thepolypeptide is at least 90% free of other proteins, lipids,carbohydrates or other materials with which it is naturally associated.In yet another embodiment, the polypeptide is at least 95% free of otherproteins, lipids, carbohydrates or other materials with which it isnaturally associated.

Conservative substitutions replace one amino acid with another aminoacid that is similar in size, hydrophobicity, etc. Examples ofconservative substitutions are shown below:

Original Residue Conservative Substitutions Ala Ser Arg Lys Asn Gln, HisAsp Glu Cys Ser Gln Asn Glu Asp His Asn; Gln Ile Leu, Val Leu Ile; ValLys Arg; Gln; Glu Met Leu; Ile Phe Met; Leu; Tyr Ser Thr Thr Ser Trp TyrTyr Trp; Phe Val Ile; Leu

Variations in the cDNA sequence that result in amino acid changes,whether conservative or not, should be minimized in order to preservethe functional and immunologic identity of the encoded protein. Thus, inseveral non-limiting examples, a Zscan4 polypeptide, or otherpolypeptides disclosed herein, includes at most two, at most five, atmost ten, at most twenty, or at most fifty conservative substitutions.The immunologic identity of the protein may be assessed by determiningwhether it is recognized by an antibody; a variant that is recognized bysuch an antibody is immunologically conserved. Any cDNA sequence variantwill preferably introduce no more than twenty, and preferably fewer thanten amino acid substitutions into the encoded polypeptide. Variant aminoacid sequences may be, for example, at least 80%, 90% or even 95% or 98%identical to the native amino acid sequence (such as a native Zscan4sequence).

Promoter: Nucleic acid control sequences which direct transcription of anucleic acid. A promoter includes necessary nucleic acid sequences nearthe start site of transcription. A promoter also optionally includesdistal enhancer or repressor elements. A “constitutive promoter” is apromoter that is continuously active and is not subject to regulation byexternal signals or molecules. In contrast, the activity of an“inducible promoter” is regulated by an external signal or molecule (forexample, a transcription factor).

Reporter gene: A gene operably linked to another gene or nucleic acidsequence of interest (such as a promoter sequence). Reporter genes areused to determine whether the gene or nucleic acid of interest isexpressed in a cell or has been activated in a cell. Reporter genestypically have easily identifiable characteristics, such asfluorescence, or easily assayed products, such as an enzyme. Reportergenes can also confer antibiotic resistance to a host cell. Exemplaryreporter genes include fluorescent and luminescent proteins (such asgreen fluorescent protein (GFP) and the red fluorescent protein from thegene dsRed), the enzyme luciferase (which catalyzes a reaction withluciferin to produce light), the lacZ gene (which encodes the proteinβ-galactosidase, which causes cells expressing the gene to appear bluewhen grown on a medium that contains the substrate analog X-gal), andthe chloramphenicol acetyltransferase (CAT) gene (which confersresistance to the antibiotic chloramphenicol). In one embodiment, thereporter gene encodes the fluorescent protein Emerald. In anotherembodiment, the reporter gene encodes the fluorescent proteinStrawberry. Additional examples are provided below.

Retinoids: A class of chemical compounds that are related chemically tovitamin A. Retinoids are used in medicine, primarily due to the way theyregulate epithelial cell growth. Retinoids have many important anddiverse functions throughout the body including roles in vision,regulation of cell proliferation and differentiation, growth of bonetissue, immune function, and activation of tumor suppressor genes.Examples of retinoids include, but are not limited to, all-transretinoic acid (atRA), 9-cis retinoic acid (9-cis RA), 13-cis RA andvitamin A (retinol).

Senescence: The inability of a cell to divide further. A senescent cellis still viable, but does not divide.

Sequence identity/similarity: The identity/similarity between two ormore nucleic acid sequences, or two or more amino acid sequences, isexpressed in terms of the identity or similarity between the sequences.Sequence identity can be measured in terms of percentage identity; thehigher the percentage, the more identical the sequences are. Sequencesimilarity can be measured in terms of percentage similarity (whichtakes into account conservative amino acid substitutions); the higherthe percentage, the more similar the sequences are. Homologs ororthologs of nucleic acid or amino acid sequences possess a relativelyhigh degree of sequence identity/similarity when aligned using standardmethods. This homology is more significant when the orthologous proteinsor cDNAs are derived from species which are more closely related (suchas human and mouse sequences), compared to species more distantlyrelated (such as human and C. elegans sequences).

Methods of alignment of sequences for comparison are well known in theart. Various programs and alignment algorithms are described in: Smith &Waterman, Adv. Appl. Math. 2:482, 1981; Needleman & Wunsch, J. Mol.Biol. 48:443, 1970; Pearson & Lipman, Proc. Natl. Acad. Sci. USA85:2444, 1988; Higgins & Sharp, Gene, 73:237-44, 1988; Higgins & Sharp,CABIOS 5:151-3, 1989; Corpet et al., Nuc. Acids Res. 16:10881-90, 1988;Huang et al. Computer Appls. in the Biosciences 8, 155-65, 1992; andPearson et al., Meth. Mol. Bio. 24:307-31, 1994. Altschul et al., J.Mol. Biol. 215:403-10, 1990, presents a detailed consideration ofsequence alignment methods and homology calculations.

The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J.Mol. Biol. 215:403-10, 1990) is available from several sources,including the National Center for Biological Information (NCBI, NationalLibrary of Medicine, Building 38A, Room 8N805, Bethesda, Md. 20894) andon the Internet, for use in connection with the sequence analysisprograms blastp, blastn, blastx, tblastn and tblastx. Additionalinformation can be found at the NCBI web site.

BLASTN is used to compare nucleic acid sequences, while BLASTP is usedto compare amino acid sequences. If the two compared sequences sharehomology, then the designated output file will present those regions ofhomology as aligned sequences. If the two compared sequences do notshare homology, then the designated output file will not present alignedsequences.

Once aligned, the number of matches is determined by counting the numberof positions where an identical nucleotide or amino acid residue ispresented in both sequences. The percent sequence identity is determinedby dividing the number of matches either by the length of the sequenceset forth in the identified sequence, or by an articulated length (suchas 100 consecutive nucleotides or amino acid residues from a sequenceset forth in an identified sequence), followed by multiplying theresulting value by 100. For example, a nucleic acid sequence that has1166 matches when aligned with a test sequence having 1154 nucleotidesis 75.0 percent identical to the test sequence (1166=1554*100=75.0). Thepercent sequence identity value is rounded to the nearest tenth. Forexample, 75.11, 75.12, 75.13, and 75.14 are rounded down to 75.1, while75.15, 75.16, 75.17, 75.18, and 75.19 are rounded up to 75.2. The lengthvalue will always be an integer. In another example, a target sequencecontaining a 20-nucleotide region that aligns with 20 consecutivenucleotides from an identified sequence as follows contains a regionthat shares 75 percent sequence identity to that identified sequence(that is, 15+20*100=75).

For comparisons of amino acid sequences of greater than about 30 aminoacids, the Blast 2 sequences function is employed using the defaultBLOSUM62 matrix set to default parameters, (gap existence cost of 11,and a per residue gap cost of 1). Homologs are typically characterizedby possession of at least 70% sequence identity counted over thefull-length alignment with an amino acid sequence using the NCBI BasicBlast 2.0, gapped blastp with databases such as the nr or swissprotdatabase. Queries searched with the blastn program are filtered withDUST (Hancock and Armstrong, 1994, Comput. Appl. Biosci. 10:67-70).Other programs may use SEG. In addition, a manual alignment can beperformed.

When aligning short peptides (fewer than around 30 amino acids), thealignment is be performed using the Blast 2 sequences function,employing the PAM30 matrix set to default parameters (open gap 9,extension gap 1 penalties). When less than the entire sequence is beingcompared for sequence identity, homologs will typically possess at least75% sequence identity over short windows of 10-20 amino acids, and canpossess sequence identities of at least 85%, 90%, 95% or 98% dependingon their identity to the reference sequence. Methods for determiningsequence identity over such short windows are described at the NCBI website.

One indication that two nucleic acid molecules are closely related isthat the two molecules hybridize to each other under stringentconditions, as described above. Nucleic acid sequences that do not showa high degree of identity may nevertheless encode identical or similar(conserved) amino acid sequences, due to the degeneracy of the geneticcode. Changes in a nucleic acid sequence can be made using thisdegeneracy to produce multiple nucleic acid molecules that all encodesubstantially the same protein. An alternative (and not necessarilycumulative) indication that two nucleic acid sequences are substantiallyidentical is that the polypeptide which the first nucleic acid encodesis immunologically cross reactive with the polypeptide encoded by thesecond nucleic acid.

One of skill in the art will appreciate that the particular sequenceidentity ranges are provided for guidance only; it is possible thatstrongly significant homologs could be obtained that fall outside theranges provided.

Stem cell: A cell having the unique capacity to produce unaltereddaughter cells (self-renewal; cell division produces at least onedaughter cell that is identical to the parent cell) and to give rise tospecialized cell types (potency). Stem cells include, but are notlimited to, ES cells, EG cells, GS cells, MAPCs, maGSCs, USSCs and adultstem cells. In one embodiment, stem cells can generate a fullydifferentiated functional cell of more than one given cell type. Therole of stem cells in vivo is to replace cells that are destroyed duringthe normal life of an animal. Generally, stem cells can divide withoutlimit. After division, the stem cell may remain as a stem cell, become aprecursor cell, or proceed to terminal differentiation. A precursor cellis a cell that can generate a fully differentiated functional cell of atleast one given cell type. Generally, precursor cells can divide. Afterdivision, a precursor cell can remain a precursor cell, or may proceedto terminal differentiation.

Subpopulation: An identifiable portion of a population. As used herein,a “subpopulation” of ES cells expressing Zscan4 is the portion of EScells in a given population that has been identified as expressingZscan4. In one embodiment, the subpopulation is identified using anexpression vector comprising a Zscan4 promoter and a reporter gene,wherein detection of expression of the reporter gene in a cell indicatesthe cell expresses Zscan4 and is part of the subpopulation.

Telomere: Refers to the end of a eukaryotic chromosome, a specializedstructure involved in the replication and stability of the chromosome.Telomeres consist of many repeats of a short DNA sequence in a specificorientation.

Telomere functions include protecting the ends of the chromosome so thatchromosomes do not end up joined together, and allowing replication ofthe extreme ends of the chromosomes (by telomerase). The number ofrepeats of telomeric DNA at the end of a chromosome decreases with age.

Therapeutic amount: An amount of a therapeutic agent sufficient toachieve the intended purpose. For example, a therapeutic amount ofZscan4⁺ ES cells is an amount sufficient to reduce a disorder orsymptoms of a disorder that can benefit from ES cell therapy. Atherapeutic amount may in some example not treat the disorder orsymptoms 100%. However, a decrease in any known feature or symptom of adisorder that can benefit from ES cell therapy (such as Zscan4⁺ EScells), such as a decrease of at least 10%, at least 15%, at least 25%,at least 30%, at least 50%, at least 60%, at least 70%, at least 75%, atleast 85%, at least 95%, or greater, can be therapeutic. The therapeuticamount of a given therapeutic agent will vary with factors such as thenature of the agent, the route of administration, the size and speciesof the animal to receive the therapeutic agent, and the purpose of theadministration. The therapeutic amount in each individual case can bedetermined empirically without undue experimentation by a skilledartisan according to established methods in the art.

Totipotent cell: Refers to a cell that can form an entire organismautonomously. Only a fertilized egg (oocyte) possesses this ability(stem cells do not).

Transfecting or transfection: Refers to the process of introducingnucleic acid into a cell or tissue. Transfection can be achieved by anyone of a number of methods, such as, but not limited to,liposomal-mediated transfection, electroporation and injection.

Vector: A nucleic acid molecule as introduced into a host cell, therebyproducing a transformed host cell. A vector may include nucleic acidsequences that permit it to replicate in a host cell, such as an originof replication (DNA sequences that participate in initiating DNAsynthesis). For example, an expression vector contains the necessaryregulatory sequences to allow transcription and translation of insertedgene or genes. A vector may also include one or more selectable markergenes and other genetic elements known in the art. Vectors include, forexample, virus vectors and plasmid vectors.

Zscan4: A group of genes identified as exhibiting 2-cell-specificexpression and ES cell-specific expression. In the mouse, the term“Zscan4” refers to a collection of genes including three pseudogenes(Zscanl-ps1, Zscan4-ps2 and Zscan4-ps3) and six expressed genes(Zscan4a, Zscan4b, Zscan4c, Zscan4d, Zscan4e and Zscan4f). As usedherein, Zscan4 also includes human ZSCAN4. Zscan4 refers to Zscan4polypeptides and Zscan4 polynucleotides encoding the Zscan4polypeptides.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. The singular terms“a,” “an,” and “the” include plural referents unless context clearlyindicates otherwise. Similarly, the word “or” is intended to include“and” unless the context clearly indicates otherwise. Hence “comprisingA or B” means including A, or B, or A and B. It is further to beunderstood that all base sizes or amino acid sizes, and all molecularweight or molecular mass values, given for nucleic acids or polypeptidesare approximate, and are provided for description. Although methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, suitable methods andmaterials are described below. All publications, patent applications,patents, GenBank Accession numbers and other references mentioned hereinare incorporated by reference in their entirety. In case of conflict,the present specification, including explanations of terms, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

III. Overview of Several Embodiments

Exceptional genomic stability is one of the hallmarks of mouse embryonicstem (ES) cells. However, the genes contributing to this stabilityremain unidentified. It is shown herein that Zscan4 is involved intelomere maintenance and long-term-genomic stability in ES cells. In thestandard ES cell culture condition that maintains the undifferentiatedstate of ES cells (e.g., including LIF), only about 5% of ES cellsexpress Zscan4 at a given time, but nearly all ES cells experience theZscane state within nine passages. The transient Zscan4-positive stateis associated with rapid telomere extension by telomere recombinationand upregulation of meiosis-specific homologous recombination genes,which encode proteins that are colocalized with ZSCAN4 on telomeres.Furthermore, Zscan4 knockdown gradually shortens telomeres, increaseskaryotype abnormalities and spontaneous sister chromatid exchange, andslows down cell proliferation until reaching crisis by eight passages.

A Zscan4 gene cluster includes 6 transcribed paralogous genes(Zscan4a-Zscan4f), which share high sequence similarities and are thuscollectively called Zscan4. A sharp expression peak of Zscan4 marks thelate 2-cell stage of mouse embryos and is essential for embryoimplantation and blastocyst outgrowth in tissue culture. Zscan4d istranscribed predominantly in 2-cell embryos, whereas Zscan4c istranscribed predominantly in ES cells and is associated with selfrenewal. Both Zscan4c and Zscan4d encode a SCAN domain, predicted tomediate protein-protein interactions, and four DNA binding Zinc-fingerdomains. It is shown herein that Zscan4 is involved in long-termmaintenance of karyotype integrity and mediation of regulated telomererecombination in normal undifferentiated ES cells.

It is also shown herein that Zscan4 has a unique function as anactivator of spontaneous telomere sister chromatic exchange (T-SCE) inundifferentiated ES cells and is involved in the regulation of telomerelength and karyotype stability (FIG. 7D). As Zscan4 knockdown eventuallyleads to karyotype deterioration and the reduction of cellproliferation, these results indicate that Zscan4 is important forlong-term self-renewal of ES cells. Furthermore, the data indicate thatkaryotype deterioration is due to telomere degradation as well as anincrease in spontaneous non-telomeric SCE. Thus, this disclosureprovides a first link to a novel mechanism employed by ES cells tosustain long-term genomic stability and telomere maintenance.

Regulation of telomeres by Zscan4 in mouse ES cells is distinctive fromthose previously reported. First, it is shown herein that Zscan4 isexpressed transiently in ES cells, and thus, at any given time only ˜5%of the undifferentiated ES cells are Zscan4 positive, whereas otherheterogeneously expressed genes in ES cells (Tanaka, Reprod. Fertil.Dev. 21: 67-75, 2009; Carter et al., Gene Expr. Patterns 8:181-198,2008) often mark specific cell lineages (Toyooka et al., Development135:909-18, 2008; Hayashi et al., Cell Stem Cell 3:391-401, 2008).Constitutive Zscan4 expression may lead to abnormally long telomeres,which may explain why this gene is not ubiquitously expressed. Indeed,the results herein show that ZSCAN4 protein is able to form foci ontelomeres along with meiosis-specific homologous recombination mediatorsindicating ES cells are able to utilize a novel mechanism for T-SCE.Second, telomere elongation by T-SCE has previously been observed incells with little or no telomerase activity, for example, in long-termcultures of telomerase knockout Terc−/− ES cells (Wang et al., Proc.Natl. Acad. Sci. USA 102:10256-60, 2005; Niida, et al., Mol. Cell. Biol.20:4115-27, 2000; Bailey et al., Nucleic Acids Res 32:3743-51, 2004).Similarly, T-SCE usually occurs in tumor cells that show no reactivationof telomerase. By contrast, it is shown herein that telomerase is activein normal undifferentiated ES cells expressing Zscan4. Third, most genespreviously identified for telomere regulation are inhibitors of T-SCE,as downregulation of these genes increases T-SCE and/or telomere length(De Boeck et al., J. Pathol. 217:327-44, 2009), such as DNAmethyltransferase DNMT1 and DNMT3 (Gonzalo et al., Nat. Cell Biol.8:416-424, 2006) and Werner syndrome protein (WRN) (Laud et al., GenesDev. 19:2560-70, 2005). An exception is the Rte1 gene, as Rte1−/− EScells show telomere shortening after induction of differentiation (Dinget al., Cell 117:873-86, 2004), but unlike Rte1, Zscan4 exhibits thisphenotype in undifferentiated ES cells. Fourth, telomere elongationthrough T-SCE usually results from general chromosomal instability,along with increased SCE in non-telomeric sequences (Wang et al., Proc.Natl. Acad. Sci. USA 102:10256-60, 2005). By contrast, T-SCE mediated byZscan4 is not associated with an increase of general SCE, and normalkaryotype remains stable with lower spontaneous SCE rate.

The expression level of Zscan4 is varied among different pluripotentstem cells, which may be correlated to their difference in genomicstability. For example, consistent with the inferior ability ofembryonal carcinoma (EC) cells to maintain genomic integrity (Blellochet al., Proc. Natl. Acad. Sci. USA 101:13985-90, 2004), the expressionof Zscan4 is much lower in EC cells than in ES cells (Aiba et al., DNARes. 16:73-80, 2009). The expression level of Zscan4 in iPS cells(Takahashi & Yamanaka, Cell 126:663-76, 2006) is comparable to ES cells(Aiba et al., DNA Res. 16:73-80, 2009), indicating that iPS may haveregained the ability of ES-like genome maintenance. By selecting cellsable to activate Zscan4, cultures enriched for cells more suitable forfuture therapeutic purposes can be generated. Moreover, inducing andcontrolling Zscan4 expression provides a means to increase the genomicstability in other cell types, such as stem cells or cancer cells.

Based on these results, provided herein are methods of increasing genomestability of an isolated ES cell or iPS cell, increasing telomere lengthin an ES or iPS cell, or both. In particular examples, such methodsinclude contacting the ES or iPS cell with an agent that increasesexpression of Zscan4 in the ES or iPS cell relative to expression ofZscan4 in an ES or iPS cell in the absence of the agent. For example,the ES or iPS cell can be incubated with the agent under conditions thatpermit the agent to enter the ES or iPS cell an increase Zscan4expression.

Further provided is a method of inducing differentiation of isolated EScells or isolated iPS cells into germ cells. In some embodiments, themethod includes contacting the ES or iPS cells with an agent thatincreases expression of Zscan4 in the ES or iPS cells, thereby inducingdifferentiation of the ES or iPS cells into germ cells. In someembodiments, the agent is a nucleic acid molecule encoding Zscan4, or aZscan4 protein or functional fragment thereof. In other embodiments, theagent is a retinoid, such as, but not limited to, atRA, 9-cis RA, 13-cisRA and vitamin A. In other embodiments, the agent induces oxidativestress.

A method of inducing meiosis, meiosis-specific recombination and/or DNArepair in an isolated ES cell or an isolated iPS cell is also provided.In some embodiments, the method includes contacting the ES or iPS cellwith an agent that increases expression of Zscan4 in the ES or iPS cell,thereby inducing meiosis, meiosis-specific recombination and/or DNArepair in the ES or iPS cell. In some embodiments, the agent is anucleic acid molecule encoding Zscan4, or a Zscan4 protein or functionalfragment thereof. In other embodiments, the agent is a retinoid, suchas, but not limited to, atRA, 9-cis RA, 13-cis RA and vitamin A. Inother embodiments, the agent induces oxidative stress.

A method of protecting a cell from a DNA-damaging agent, comprisingcontacting the cell with an agent that increases expression of Zscan4and exposing the cell to a DNA-damaging agent, wherein an increase insurvival of the cell relative to a control indicates the agent protectsthe cell from the DNA-damaging agent. In some embodiments, the controlis a cell exposed to the DNA-damaging agent that has not been induced toexpress Zscan4. In some embodiments, the cell is contacted with theagent prior to being exposed to the DNA-damaging agent. In otherembodiments, the cell is contacted with the agent while simultaneouslyexposed to the DNA-damaging agent. In yet other embodiments, the cell iscontacted with the agent after being exposed to the DNA-damaging agent.In some embodiments, the agent is a nucleic acid molecule encodingZscan4, or a Zscan4 protein or functional fragment thereof. In someembodiments, the agent is a retinoid, such as, but not limited to, atRA,9-cis RA, 13-cis RA and vitamin A. In some embodiments, the agentinduces oxidative stress. In some embodiments, the DNA-damaging agent isa chemotherapeutic drug. In particular examples, the DNA-damaging agentis mitomycin C or cisplatin.

It is noted that although ES cells are described throughout theapplication, one skilled in the art will appreciate that iPS cells canbe used instead of the ES cells in the disclosed compositions andmethods. Thus, for example, methods of increasing genome stability of aniPS cell, increasing telomere length in an iPS cell, or both, areprovided, as are methods of using such cells to treat patients in needof ES cell therapy. iPS cells are very similar to ES cells, but can bemade from human fibroblast and other differentiated cells without goingthrough the nuclear transplantation (cloning) procedure. Zscan4 isexpressed at the same level in iPS cells as in the ES cells, and theinventors have shown that Zscan4 is also expressed only a small fraction(5%) of iPS cells in culture. Thus, human or other mammalian iPS cellscan be used in place of (or in addition to) ES cells in the methodsprovided herein, such as iPS cells expressing Zscan4 (Zscane iPS cells).

Moreover, it is further noted that the disclosed compositions andmethods can be useful for the treatment of cancer (described in furtherdetail below). Thus, although ES cells are described throughout theapplication, one skilled in the art will appreciate that cancer cellscan be used instead of the ES cells in the disclosed compositions andmethods. For example, methods of increasing genome stability of a cancercell, increasing telomere length in a cancer cell, or both, areprovided, as are methods of treating a subject with cancer byadministering a Zscan4 polypeptide or polynucleotide, such asadministration directly to cancer cells.

Exemplary agents that can increase Zscan4 expression in a cell includeisolated nucleic acid molecules encoding Zscan4. Zscan4 protein andcoding sequences are well-known in the art as discussed in detail below.Any of the molecules described in section IV below can be used in themethods provided herein. One skilled in the art will appreciate that anyZscan4 coding sequence can be used, such as a mouse Zscan4c- or humanZSCAN⁴-encoding nucleic acid sequence. For example, ES or iPS cells canbe transfected with a Zscan4-encoding isolated nucleic acid moleculeunder conditions sufficient to allow for expression of Zscan4 in the ESor iPS cell. In some examples, the isolated nucleic acid moleculesencoding Zscan4 are part of a vector, such as a viral or plasmid vector.In one example, the isolated nucleic acid molecule encoding Zscan4 canbe operably linked to a promoter that drives expression of Zcsan4.Constitutive and inducible promoters can be used.

In some embodiments, the agent that induces Zscan4 expression is aretinoid. Exemplary retinoids include, but are not limited to atRA,9-cis RA, 13-cis RA and vitamin A. In other embodiments, the agent thatinduces Zscan4 expression is an agent that induces oxidative stress, forexample hydrogen peroxide.

Also provided are methods for increasing genome stability in apopulation of ES or iPS cells, increasing telomere length in apopulation of ES or iPS cells, or both. In particular examples, themethod includes selecting Zscan4⁺ ES or iPS cells from the population ofES or iPS cells. That is, a population of ES or iPS cells containing ESor iPS cells expressing Zscan4 and ES or iPS cells not expressing Zscan4can be enriched for the Zscan4-expressing ES or iPS cells for example byeliminating the non-Zscan4-expressing cells or selecting for theZscan4-expressing cells. In one example, Zscane cells are selected bytransfecting the population of cells with an expression vector thatincludes at least a Zscan4 promoter and a reporter gene, whereinexpression of the reporter gene indicates Zscan4 is expressed in thesubpopulation of ES or iPS cells. The cells in which Zscan4 expressionis detected can be selected, for example by FACS. In one example, theexpression vector is the nucleic acid sequence set forth as SEQ ID NO:38, or a sequence having at least 80%, at least 90%, at least 95%, or atleast 98% sequence identity to SEQ ID NO: 38. In another example, thereporter gene is a drug (e.g., antibiotic)-selectable marker, whereinthe non-Zscan4-expressing cells are killed by adding the appropriatedrug (e.g., hygromycin, neomycin, etc).

Methods are also provided for treating a subject in need of ES celltherapy. In some examples, the method includes selecting a subject inneed of treatment and administering to the selected subject asubpopulation of undifferentiated ES (or iPS) cells that are Zscan4⁺.Examples of subjects that can benefit from ES cell therapy include butare not limited to subjects having cancer, an autoimmune disease,neurologic injury or a neurodegenerative disorder, as well as otherdisorders where cellular regeneration is desired (such as wound healing,muscle repair (including cardiac), cartilage replacement (e.g., to treatarthritis), tooth regeneration, blindness, deafness, bone marrowtransplant, and Crohn's disease). In some examples, the method includesselecting Zscan4⁺ ES or iPS cells from a population of ES or iPS cells,and the Zscan4⁺ ES or iPS cells are administered to the subject. Forexample, Zscan4⁺ ES or iPS cells can be selected by transfecting thepopulation of ES or iPS cells with an expression vector that includes aZscan4 promoter (such as a Zscan4c promoter) and a reporter gene,wherein expression of the reporter gene indicates Zscan4 is expressed inthe subpopulation of ES or iPS cells.

Also provided herein is a method of treating a subject with cancer byadministering to the subject a Zscan4 polypeptide or polynucleotide. Insome embodiments, the method further includes selecting a patient inneed of such therapy. In some embodiments, the method includesadministering the Zscan4 polypeptide or polynucleotide directly to tumorcells to tumor tissue, such as by injection. In particular examples, thesubject is administered a vector comprising a Zscan4 polynucleotide. Inother embodiments, the Zscan4 polypeptide is encapsulated by ananoparticle.

Further provided is a method of enhancing chemoresponsiveness of a tumorin a subject, comprising administering to the subject an agent thatinhibits expression of Zscan4. In some examples, the agent isadministered directly to the tumor cells. A method of increasing theefficacy of a chemotherapeutic agent in an isolated cell by contactingthe cell with an agent that inhibits expression of Zscan4 is alsoprovided.

IV. Zscan4 Polynucleotide and Polypeptide Sequences

Zscan4 nucleic acid and amino acid sequences have been previouslydescribed in the art (see, for example, WO 2008/118957, the disclosureof which is herein incorporated by reference; Falco et al., Dev. Biol.307(2):539-550, 2007; and Carter et al., Gene Expr. Patterns.8(3):181-198, 2008). As used herein, the term “Zscan4” includes any oneof a group of mouse genes exhibiting 2-cell embryonic stage- or EScell-specific expression (including Zscan4a, Zscan4b, Zscan4c, Zscan4d,Zscan4e and Zscan4f), the human ortholog ZSCAN4, or any other speciesortholog of ZSCAN4. In a specific example, Zscan4 is mouse Zscan4c orhuman ZSCAN4.

Exemplary Zscan4 amino acid sequences are set forth in the SequenceListing as SEQ ID NO: 25 (Zscan4a), SEQ ID NO: 27 (Zscan4b), SEQ ID NO:29 (Zscan4c), SEQ ID NO: 31 (Zscan4d), SEQ ID NO: 33 (Zscan4e), SEQ IDNO: 35 (Zscan4f) and SEQ ID NO: 37 (human ZSCAN4). One skilled in theart will appreciate that sequences having at least 80%, at least 90%, atleast 95%, or at least 98% sequence identity to these sequences andretain Zscan4 activity (such as the ability to enhance genome stabilityand increase telomere length in a ES cell) can be used in the methodsprovided herein.

ZSCAN4 amino acid sequences from other species are publically available,including dog ZSCAN4 (GenBank Accession Nos. XP_(—)541370.2 andXP_(—)853650.1); cow ZSCAN4 (GenBank Accession No. XP_(—)001789302.1);and horse ZSCAN4 (GenBank Accession No. XP_(—)001493994.1). Each of theabove-listed GenBank Accession numbers is herein incorporated byreferences as it appears in the GenBank database on Sep. 4, 2009.

Specific, non-limiting examples of Zscan4 polypeptides that can beexpressed in ES cells using the methods provided herein includepolypeptides including an amino acid sequence at least 80%, at least85%, at least 90%, at least 95%, at least 96%, at least 97%, at least98% or at least 99% homologous to the amino acid sequence set forth inSEQ ID NO: 25, 27, 29, 31, 33, 35 or 37. In a further embodiment, aZscan4 polypeptide is a conservative variant of SEQ ID NO: 25, 27, 29,31, 33, 35 or 37, such that it includes no more than fifty conservativeamino acid substitutions, such as no more than two, no more than five,no more than ten, no more than twenty, or no more than fiftyconservative amino acid substitutions in SEQ ID NO: 25, 27, 29, 31, 33,35 or 37. In another embodiment, a Zscan4 polypeptide has an amino acidsequence comprising the amino acid sequence set forth in SEQ ID NO: 25,27, 29, 31, 33, 35 or 37. In another embodiment, a Zscan4 polypeptidehas an amino acid sequence consisting of the amino acid sequence setforth in SEQ ID NO: 25, 27, 29, 31, 33, 35 or 37.

Fragments and variants of a Zscan4 polypeptide can readily be preparedby one of skill in the art using molecular techniques. In oneembodiment, a fragment of a Zscan4 polypeptide includes at least 50, atleast 100, at least 150, at least 200, at least 250, at least 300, atleast 350, at least 400, at least 450 or at least 500 consecutive aminoacids of the Zscan4 polypeptide. In a further embodiment, a fragment ofZscan4 is a fragment that confers a function of Zscan4 when transferredinto a cell of interest, such as, but not limited to, enhancing genomestability and/or increasing telomere length.

Minor modifications of the Zscan4 polypeptide primary amino acidsequences may result in peptides which have substantially equivalentactivity as compared to the unmodified counterpart polypeptide describedherein. Such modifications may be deliberate, as by site-directedmutagenesis, or may be spontaneous. All of the polypeptides produced bythese modifications are included herein.

One of skill in the art can readily produce fusion proteins including aZscan4 polypeptide and a second polypeptide of interest. Optionally, alinker can be included between the Zscan4 polypeptide and the secondpolypeptide of interest. Fusion proteins include, but are not limitedto, a polypeptide including a Zscan4 polypeptide and a marker protein.In one embodiment, the marker protein can be used to identify or purifya Zscan4 polypeptide. Exemplary fusion proteins include, but are notlimited to, green fluorescent protein, six histidine residues, or mycand a Zscan4 polypeptide.

One skilled in the art will appreciate that such variants, fragments,and fusions of Zscan4 useful for the disclosed methods are those thatretain Zscan4 activity (such as the ability to enhance genome stabilityand increase telomere length or both in an ES cell).

Nucleic acid molecules encoding a Zscan4 polypeptide are termed Zscan4polynucleotides or nucleic acid molecules. These polynucleotides includeDNA, cDNA and RNA sequences which encode a Zscan4. It is understood thatall polynucleotides encoding a Zscan4 polypeptide are also includedherein, as long as they encode a polypeptide with a recognized Zscan4activity, such as the ability to modulate genome stability or telomerelength. The polynucleotides include sequences that are degenerate as aresult of the genetic code. There are 20 natural amino acids, most ofwhich are specified by more than one codon. Therefore, all degeneratenucleotide sequences are included as long as the amino acid sequence ofthe Zscan4 polypeptide encoded by the nucleotide sequence isfunctionally unchanged. A Zscan4 polynucleotide encodes a Zscan4polypeptide, as disclosed herein. Exemplary polynucleotide sequencesencoding Zscan4 that can be expressed in ES or iPS cells using themethods provided herein are set forth in the Sequence Listing as SEQ IDNO: 24 (Zscan4a), SEQ ID NO: 26 (Zscan4b), SEQ ID NO: 28 (Zscan4c), SEQID NO: 30 (Zscan4d), SEQ ID NO: 32 (Zscan4e), SEQ ID NO: 34 (Zscan4f)and SEQ ID NO: 36 (human ZSCAN4).

ZSCAN4 nucleic acid sequences from other species are publicallyavailable, including dog ZSCAN4 (GenBank Accession Nos. XM_(—)541370.2and XM_(—)848557.1); cow ZSCAN4 (GenBank Accession No.XM_(—)001789250.1); and horse ZSCAN4 (GenBank Accession No.XM_(—)001493944.1). Each of the above-listed GenBank Accession numbersis herein incorporated by references as it appears in the GenBankdatabase on Aug. 11, 2009.

In some embodiments, the Zscan4 polynucleotide sequence expressed in anES or iPS cell using the methods provided herein is at least 80%, atleast 85%, at least 90%, at least 95%, at least 96%, at least 97%, atleast 98% or at least 99% identical to SEQ ID NO: 24, 26, 28, 30, 32, 34or 36. In some embodiments, the Zscan4 polynucleotide sequence comprisesthe nucleic acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32, 34or 36. In some embodiments, the Zscan4 polynucleotide sequence consistsof the nucleic acid sequence set forth in SEQ ID NO: 24, 26, 28, 30, 32,34 or 36. In particular examples, the Zscan4 polynucleotide sequenceexpressed in an ES or iPS cell is exogenous to the cell. For example,the Zscan4 polynucleotide sequence can be a recombinant or non-nativesequence to the ES or iPS cell.

Fragments and variants of Zscan4 polynucleotides can readily be preparedby one of skill in the art using molecular techniques. In oneembodiment, a fragment of a Zscan4 polynucleotide includes at least 250,at least 500, at least 750, at least 1000, at least 1500, or at least2000 consecutive nucleic acids of the Zscan4 polynucleotide. In afurther embodiment, a fragment of Zscan4 is a fragment that confers afunction of Zscan4 when expressed in a cell of interest, such as, butnot limited to, enhancing genome stability and/or increasing telomerelength.

Minor modifications of the Zscan4 polynucleotide sequences may result inexpression of peptides which have substantially equivalent activity ascompared to the unmodified counterpart polynucleotides described herein.Such modifications may be deliberate, as by site-directed mutagenesis,or may be spontaneous. All of the polynucleotides produced by thesemodifications are included herein.

Zscan4 polynucleotides include recombinant DNA which is incorporatedinto a vector; into an autonomously replicating plasmid or virus; orinto the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g., a cDNA) independent of other sequences. Thenucleotides can be ribonucleotides, deoxyribonucleotides, or modifiedforms of either nucleotide. The term includes single- anddouble-stranded forms of DNA.

With the provision of several Zscan4 nucleic acid and protein sequencesdescribed above, the expression of any Zscan4 protein (e.g., aheterologous Zscan4 protein) in an ES or iPS cell using standardlaboratory techniques is now enabled. In some examples, the Zscan4nucleic acid sequence is under the control of a promoter. In someexamples, a vector system is used to express Zscan4, such as plasmids,bacteriophages, cosmids, animal viruses and yeast artificial chromosomes(YACs). These vectors may then be introduced into ES or iPS cells, whichare rendered recombinant by the introduction of the heterologous Zscan4cDNA.

A Zscan4 coding sequence may be operably linked to a heterologouspromoter, to direct transcription of the Zscan4 coding nucleic acidsequence. A promoter includes necessary nucleic acid sequences near thestart site of transcription, such as, in the case of a polymerase IItype promoter, a TATA element. A promoter also optionally includesdistal enhancer or repressor elements which can be located as much asseveral thousand base pairs from the start site of transcription. In oneexample, the promoter is a constitutive promoter, such as theCAG-promoter (Niwa et al., Gene 108(2):193-9, 1991), or thephosphoglycerate kinase (PGK)-promoter. In another example, the promoteris an inducible promoter such as a tetracycline-inducible promoter(Masui et al., Nucleic Acids Res. 33:e43, 2005). Other exemplarypromoters that can be used to drive Zscan4 expression include but arenot limited to: lac system, the trp system, the tac system, the trcsystem, major operator and promoter regions of phage lambda, the controlregion of fd coat protein, the early and late promoters of SV40,promoters derived from polyoma, adenovirus, retrovirus, baculovirus andsimian virus, the promoter for 3-phosphoglycerate kinase, the promotersof yeast acid phosphatase, and the promoter of the yeast alpha-matingfactors. In some examples, a native Zscan4 promoter is used.

A vector system can used to express Zscan4. Exemplary vectors that canbe used to express Zscan4 in ES cells include but are not limited toplasmids and viral vectors. In one example, vectors containing thepromoter and enhancer regions of the SV40 or long terminal repeat (LTR)of the Rous Sarcoma virus and polyadenylation and splicing signal fromSV40 (Mulligan and Berg, 1981, Proc. Natl. Acad. Sci. USA 78:2072-6;Gorman et al., 1982, Proc. Natl. Acad. Sci. USA 78:6777-81) are used. Inone example, the vector is a viral vector, such as an adenoviral vector,an adeno-associated virus (AAV), such as described in U.S. Pat. No.4,797,368 (Carter et al.) and in McLaughlin et al. (J. Virol.62:1963-73, 1988) and AAV type 4 (Chiorini et al. J. Virol. 71:6823-33,1997) and AAV type 5 (Chiorini et al. J. Virol. 73:1309-19, 1999), orretroviral vector (such as the Moloney Murine Leukemia Virus, spleennecrosis virus, and vectors derived from retroviruses such as RousSarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, humanimmunodeficiency virus, myeloproliferative sarcoma virus, and mammarytumor virus). Other viral transfection systems may also be utilized,including Vaccinia virus (Moss et al., 1987, Annu. Rev. Immunol.5:305-24), Bovine Papilloma virus (Rasmussen et al., 1987, MethodsEnzymol. 139:642-54) or members of the herpes virus group such asEpstein-Barr virus (Margolskee et al., 1988, Mol. Cell. Biol.8:2837-47). In addition, vectors may contain antibiotic selectablemarkers (such as neomycin, hygromycin or mycophoenolic acid) to permitselection of transfected cells that exhibit stable, long-term expressionof the vectors (and therefore the Zscan4 nucleic acid).

The vectors can be maintained in the cells as episomal, freelyreplicating entities by using regulatory elements of viruses such aspapilloma (Sarver et al., 1981, Mol. Cell. Biol. 1:486) or Epstein-Barr(Sugden et al., 1985, Mol. Cell. Biol. 5:410). Alternatively, one canalso produce cell lines that have integrated the vector into genomicDNA. Both of these types of cell lines produce the gene product on acontinuous basis. One can also produce cell lines that have amplifiedthe number of copies of the vector (and therefore of the cDNA as well)to create cell lines that can produce high levels of the gene product.

The transfer of DNA into human or other mammalian cells is aconventional technique. For example, an isolated Zscan4 nucleic acidsequence (for example as a naked DNA or as part of an expression vector)can be introduced into the recipient cells for example by precipitationwith calcium phosphate (Graham and vander Eb, 1973, Virology 52:466) orstrontium phosphate (Brash et al., 1987, Mol. Cell. Biol. 7:2013),electroporation (Neumann et al., 1982, EMBO J. 1:841), lipofection(Felgner et al., 1987, Proc. Natl. Acad. Sci. USA 84:7413), DEAE dextran(McCuthan et al., 1968, J. Natl. Cancer Inst. 41:351), microinjection(Mueller et al., 1978, Cell 15:579), protoplast fusion (Schafner, 1980,Proc. Natl. Acad. Sci. USA 77:2163-7), or pellet guns (Klein et al.,1987, Nature 327:70). Alternatively, the Zscan4 nucleic acid sequencecan be part of a viral vector, which is introduced into the ES cells byinfection. Systems are developed that use, for example, retroviruses(Bernstein et al., 1985, Gen. Engrg. 7:235), adenoviruses (Ahmad et al.,1986, J. Virol. 57:267), or Herpes virus (Spaete et al., 1982, Cell30:295).

V. Measuring Genome Stability

Methods are provided for increasing genome stability and/or increasingtelomere length in ES cells or a population of ES cells (such as Zscan4⁺ES cells). In particular examples, genome stability is increased in anES cell by at least 20%, at least 40%, at least 50%, at least 60%, atleast 75%, at least 80%, at least 90%, at least 95%, or at least 98%,for example relative to an ES cell not expressing Zscan4 (or value orrange of values expected in a Zscan4⁻ ES cell) or relative to mouseembryo fibroblast (MEF) cells or skin fibroblast cells. Methods ofmeasuring genome stability and telomere length are routine in the art,and the disclosure is not limited to particular methods. The particularexamples provided herein are exemplary.

In some examples, genome stability in an ES cell, such as a Zscan4⁺ EScell, is measured by detecting cell proliferation. Genome stability isincreased if cell proliferation is increased, for example relative toZscan4⁻ ES cells. For example, ES cell proliferation can be detected bygrowing ES cells in culture and measuring the doubling time of the cellsafter each passage. In one example, genome stability is increased ifcrisis (e.g., cell death) does not occur at passage 8 or earlier.

In some examples, genome stability in an ES cell, such as a Zscan4⁺ EScell, is measured by performing karyotype analysis. Genome stability isincreased if the presence of karyotype abnormalities (such as chromosomefusions and fragmentations) is decreased or even absent, for examplerelative to Zscan4⁻ ES cells. For example, karyotype analysis can beperformed in an ES cell by inducing metaphase arrests, then preparingmetaphase chromosome spreads.

In some examples, genome stability in an ES cell, such as a Zscan4⁺ EScell, is measured by measuring telomere sister chromatid exchange(T-SCE). Genome stability is increased if the presence of T-SCE isincreased, for example relative to Zscan4⁻ ES cells. For example, T-SCEcan be measured in an ES cell by using telomere chromosome-orientationFISH(CO-FISH).

In some examples, genome stability in an ES cell, such as a Zscan4⁺ EScell, is measured by measuring sister chromatid exchange (SCE). Genomestability is increased if the presence of SCE is decreased, for examplerelative to Zscan4⁻ ES cells. For example, SCE can be measured in an EScell by detecting SCE in a metaphase spread.

In some examples, telomere length is measured in an ES cell, such as aZscan4⁺ ES cell. In some examples, telomere length is increased in an EScell if the length of the telomeres is greater, for example relative totelomere length in Zscan4⁻ ES cells. For example, telomere length can bedetected in an ES cell by fluorescence in situ hybridization (FISH),quantitative FISH (Q-FISH), or telomere qPCR.

VI. Zscan4 Promoter Sequences and Expression Vectors

In particular examples, genome stability and/or telomere length in apopulation of ES cells is increased by selecting Zscan4⁺ ES cells fromthe population of ES cells. For example, an expression vector comprisinga Zsan4 promoter sequence operably linked to a nucleic acid sequenceencoding a heterologous polypeptide (such as a reporter gene) can beused to identify cells that express Zscan4. Methods of detectingexpression of the reporter gene, and thus the Zscan4+ ES cells, varydepending upon the type of reporter gene and are well known in the art.For example, when a fluorescent reporter is used, detection ofexpression can be achieved by FACS or fluorescence microscopy.Identification of a subpopulation of stem cells expressing Zscan4 can beachieved with alternative methods, including, but not limited to, usingantibodies specific for Zscan4 or by in situ hybridization.

In some examples a heterologous nucleic acid sequence (such as areporter molecule) is expressed under the control of a Zscan4 promoter(for example in a vector). In some embodiments, the Zscan4 promoter is aZscan4c promoter. For example, the Zscan4c promoter can include thenucleic acid sequence set forth as nucleotides 906-4468 of SEQ ID NO:38. In some examples, the Zscan4c promoter comprises Zscan4c exon and/orintron sequence. Other expression control sequences, includingappropriate enhancers, transcription terminators, a start codon (i.e.,ATG) in front of a protein-encoding gene, splicing signals for introns,and stop codons can be included with the Zscan4 promoter in anexpression vector. Generally the promoter includes at least a minimalsequence sufficient to direct transcription of a heterologous nucleicacid sequence. In several examples, the heterologous nucleic acidsequence encodes a reporter molecule.

In other examples, a heterologous nucleic acid sequence (such as areporter molecule) is incorporated into a subject's genomic DNA, such asby homologous recombination. For example, the coding sequence for GFPcould be inserted into the coding region of ZSCAN4, or could replace thecoding region of ZSCAN4, ushc that GFP is expressed in the same manneras endogenous Zscan4. Gene “knock-in” methods by homologousrecombination are well known in the art.

The heterologous protein encoded by the heterologous nucleic acidsequence is typically a reporter molecule, such as a marker, an enzyme,a fluorescent protein, a polypeptide that confers antibiotic resistanceto the cell or an antigen that can be identified using conventionalmolecular biology procedures. Reporter molecules can be used to identifya cell, or a population of cells, of interest, such as Zscanr ES cells.In one embodiment, the heterologous protein is a fluorescent marker(such as a green fluorescent protein, or a variant thereof, e.g. Emerald(Invitrogen, Carlsbad, Calif.)) an antigenic marker (such as humangrowth hormone, human insulin, human HLA antigens); a cell-surfacemarker (such as CD4, or any cell surface receptor); or an enzymaticmarker (such as lacZ, alkaline phosphatase). Expression of the reportergene indicates the cell expresses Zscan4. Methods of detectingexpression of the reporter gene vary depending upon the type of reportergene and are well known in the art. For example, when a fluorescentreporter is used, detection of expression can be achieved by FACS orfluorescence microscopy.

Expression vectors typically contain an origin of replication as well asspecific genes which allow phenotypic selection of the transformedcells, such as an antibiotic resistance gene. Vectors suitable for useare well known in the art, including viral vectors and plasmid vectors(such as those described in Section IV above). In one example, anenhancer is located upstream of the Zscan4 promoter, but enhancerelements can generally be located anywhere on the vector and still havean enhancing effect. However, the amount of increased activity willgenerally diminish with distance. Additionally, two or more copies of anenhancer sequence can be operably linked one after the other to producean even greater increase in promoter activity.

Expression vectors including a Zscan4 promoter can be used to transformhost cells, such as, but not limited to ES cells. Biologicallyfunctional viral and plasmid DNA vectors capable of expression andreplication in a host are known in the art, and can be used to transfectany cell of interest.

A “transfected cell” is a host cell into which (or into an ancestor ofwhich) has been introduced a nucleic acid molecule (e.g., DNA molecule),such as a DNA molecule including a Zscan4 promoter element. Transfectionof a host cell with a recombinant nucleic acid molecule may be carriedout by conventional techniques as are well known to those skilled in theart. As used herein, transfection includes liposomal-mediatedtransfection, electroporation, injection or any other suitable techniquefor introducing a nucleic acid molecule into a cell.

VII. Isolation of Embryonic Stem Cells

Mammalian ES cells, such as murine, primate or human ES cells, can beutilized with the methods disclosed herein. ES cells can proliferateindefinitely in an undifferentiated state. Furthermore, ES cells arepluripotent cells, meaning that they can generate all of the cellspresent in the body (bone, muscle, brain cells, etc.). ES cells havebeen isolated from the inner cell mass (ICM) of the developing murineblastocyst (Evans et al., Nature 292:154-156, 1981; Martin et al., Proc.Natl. Acad. Sci. 78:7634-7636, 1981; Robertson et al., Nature323:445-448, 1986). Additionally, human cells with ES properties havebeen isolated from the inner blastocyst cell mass (Thomson et al.,Science 282:1145-1147, 1998) and developing germ cells (Shamblott etal., Proc. Natl. Acad. Sci. USA 95:13726-13731, 1998), and human andnon-human primate embryonic stem cells have been produced (see U.S. Pat.No. 6,200,806).

As disclosed in U.S. Pat. No. 6,200,806, ES cells can be produced fromhuman and non-human primates. In one embodiment, primate ES cells arecells that express SSEA-3; SSEA-4, TRA-1-60, and TRA-1-81 (see U.S. Pat.No. 6,200,806). ES cells can be isolated, for example, using ES medium,which consists of 80% Dulbecco's modified Eagle's medium (DMEM; nopyruvate, high glucose formulation, Gibco BRL), with 20% fetal bovineserum (FBS; Hyclone), 0.1 mM B-mercaptoethanol (Sigma), 1% non-essentialamino acid stock (Gibco BRL). Generally, primate ES cells are isolatedon a confluent layer of murine embryonic fibroblast in the presence ofES cell medium. In one example, embryonic fibroblasts are obtained from12 day old fetuses from outbred mice (such as CF1, available fromSASCO), but other strains may be used as an alternative. Tissue culturedishes treated with 0.1% gelatin (type I; Sigma) can be utilized.Distinguishing features of ES cells, as compared to the committed“multipotential” stem cells present in adults, include the capacity ofES cells to maintain an undifferentiated state indefinitely in culture,and the potential that ES cells have to develop into every differentcell types. Unlike mouse ES cells, human ES (hES) cells do not expressthe stage-specific embryonic antigen SSEA-1, but express SSEA-4, whichis another glycolipid cell surface antigen recognized by a specificmonoclonal antibody (see, e.g., Amit et al., Devel. Biol. 227:271-278,2000).

For rhesus monkey embryos, adult female rhesus monkeys (greater thanfour years old) demonstrating normal ovarian cycles are observed dailyfor evidence of menstrual bleeding (day 1 of cycle=the day of onset ofmenses). Blood samples are drawn daily during the follicular phasestarting from day 8 of the menstrual cycle, and serum concentrations ofluteinizing hormone are determined by radioimmunoassay. The female ispaired with a male rhesus monkey of proven fertility from day 9 of themenstrual cycle until 48 hours after the luteinizing hormone surge;ovulation is taken as the day following the leutinizing hormone surge.Expanded blastocysts are collected by non-surgical uterine flushing atsix days after ovulation. This procedure generally results in therecovery of an average 0.4 to 0.6 viable embryos per rhesus monkey permonth (Seshagiri et al., Am J. Primatol. 29:81-91, 1993).

For marmoset embryos, adult female marmosets (greater than two years ofage) demonstrating regular ovarian cycles are maintained in familygroups, with a fertile male and up to five progeny. Ovarian cycles arecontrolled by intramuscular injection of 0.75 g of the prostaglandinPGF2a analog cloprostenol (Estrumate, Mobay Corp, Shawnee, Kans.) duringthe middle to late luteal phase. Blood samples are drawn on day 0(immediately before cloprostenol injection), and on days 3, 7, 9, 11,and 13. Plasma progesterone concentrations are determined by ELISA. Theday of ovulation is taken as the day preceding a plasma progesteroneconcentration of 10 ng/ml or more. At eight days after ovulation,expanded blastocysts are recovered by a non-surgical uterine flushprocedure (Thomson et al., J Med. Primatol. 23:333-336, 1994). Thisprocedure results in the average production of one viable embryo permarmoset per month.

The zona pellucida is removed from blastocysts, such as by briefexposure to pronase (Sigma). For immunosurgery, blastocysts are exposedto a 1:50 dilution of rabbit anti-marmoset spleen cell antiserum (formarmoset blastocysts) or a 1:50 dilution of rabbit anti-rhesus monkey(for rhesus monkey blastocysts) in DMEM for 30 minutes, then washed for5 minutes three times in DMEM, then exposed to a 1:5 dilution of Guineapig complement (Gibco) for 3 minutes. After two further washes in DMEM,lysed trophoectoderm cells are removed from the intact inner cell mass(ICM) by gentle pipetting, and the ICM plated on mouse inactivated (3000rads gamma irradiation) embryonic fibroblasts.

After 7-21 days, ICM-derived masses are removed from endoderm outgrowthswith a micropipette with direct observation under a stereo microscope,exposed to 0.05% Trypsin-EDTA (Gibco) supplemented with 1% chicken serumfor 3-5 minutes and gently dissociated by gentle pipetting through aflame polished micropipette.

Dissociated cells are re-plated on embryonic feeder layers in fresh ESmedium, and observed for colony formation. Colonies demonstratingES-like morphology are individually selected, and split again asdescribed above. The ES-like morphology is defined as compact colonieshaving a high nucleus to cytoplasm ratio and prominent nucleoli.Resulting ES cells are then routinely split by brief trypsinization orexposure to Dulbecco's Phosphate Buffered Saline (PBS, without calciumor magnesium and with 2 mM EDTA) every 1-2 weeks as the cultures becomedense. Early passage cells are also frozen and stored in liquidnitrogen.

Cell lines may be karyotyped with a standard G-banding technique (suchas by the Cytogenetics Laboratory of the University of Wisconsin StateHygiene Laboratory, which provides routine karyotyping services) andcompared to published karyotypes for the primate species.

Isolation of ES cell lines from other primate species would follow asimilar procedure, except that the rate of development to blastocyst canvary by a few days between species, and the rate of development of thecultured ICMs will vary between species. For example, six days afterovulation, rhesus monkey embryos are at the expanded blastocyst stage,whereas marmoset embryos do not reach the same stage until 7-8 daysafter ovulation. The rhesus ES cell lines can be obtained by splittingthe ICM-derived cells for the first time at 7-16 days afterimmunosurgery; whereas the marmoset ES cells were derived with theinitial split at 7-10 days after immunosurgery. Because other primatesalso vary in their developmental rate, the timing of embryo collection,and the timing of the initial ICM split, varies between primate species,but the same techniques and culture conditions will allow ES cellisolation (see U.S. Pat. No. 6,200,806 for a complete discussion ofprimate ES cells and their production).

Human ES cell lines exist and can be used in the methods disclosedherein. Human ES cells can also be derived from preimplantation embryosfrom in vitro fertilized (IVF) embryos. Only high quality embryos aresuitable for ES isolation. Present defined culture conditions forculturing the one cell human embryo to the expanded blastocyst have beendescribed (see Bongso et al., Hum Reprod. 4:706-713, 1989). Co-culturingof human embryos with human oviductal cells results in the production ofhigh blastocyst quality. IVF-derived expanded human blastocysts grown incellular co-culture, or in improved defined medium, allows isolation ofhuman ES cells with the same procedures described above for non-humanprimates (see U.S. Pat. No. 6,200,806).

VIII. Therapeutic use of Zscan4⁺ ES Cells

Methods are provided for treating subjects in need of ES cell therapy.These methods include the use of ES cells and/or iPS cells. Inparticular examples, the method includes selecting a subject in need oftreatment, and administering to the subject a therapeutic amount of asubpopulation of undifferentiated ES cells, wherein the subpopulation ofundifferentiated ES cells are Zscan4⁺. In other examples, the methodincludes administration of more mature cells (e.g., mature neuron,muscle cells, cells of a particular organ, etc.) differentiated in vitrofrom the undifferentiated ES cells (particularly a Zscan4⁺subpopulation). Cells differentiated from the Zscan4⁺ ES or iPS cellswill have better genome stability than Zscan4⁻ cells. Administration ofthe Zscan4⁺ ES cells (either undifferentiated or more mature cells)thereby treats a disease in the subject. Methods of selecting orgenerating undifferentiated ES cells that express (Zscan4⁺) aredescribed above.

Methods of differentiating undifferentiated ES cells in vitro are known.Differentiation of undifferentiated ES cells results in the formation ofcells expressing markers known to be associated with cells that are morespecialized and closer to becoming terminally differentiated cellsincapable of further division or differentiation. The pathway alongwhich cells progress from a less committed cell, to a cell that isincreasingly committed to a particular cell type, and eventually to aterminally differentiated cell is referred to as progressivedifferentiation or progressive commitment. Cells which are morespecialized (e.g., have begun to progress along a path of progressivedifferentiation) but not yet terminally differentiated are referred toas partially differentiated. For example US Patent Application No.2006/0194321 describes differentiation of ES cells into endodermal cells(e.g., pancreatic), US Patent Application No. 2004/0014209 describesdifferentiation of ES cells into cardiac cells and US Patent ApplicationNo. 2008/0194023 describes differentiation of ES cells into vascularsmooth muscle cells.

Subjects that can be treated using the methods provided herein includemammalian subjects, such as a veterinary or human subject. Subjectsinclude a fetus, newborns, infants, children, and/or adults. Inparticular examples, the subject to be treated is selected, such asselecting a subject that would benefit from ES cell therapy,particularly therapy that includes administration of Zscan4⁺ ES cells.

Examples of disorders or diseases that can benefit from administrationof ES cells, such as Zscan4⁺ ES cells include, cancer, autoimmunediseases, and diseases in which cell regeneration is beneficial, such asneurologic injuries or a neurodegenerative disorders, as well asblindness, deafness, tooth loss, arthritis, myocardial infarctions, bonemarrow transplants, baldness, Crohn's disease, diabetes, and musculardystrophy. In particular examples, a subject having one or more of thesedisorders is selected for the treatments herein disclosed.

Cancers include malignant tumors that are characterized by abnormal oruncontrolled cell growth. Patients treated with the ES cells disclosedherein may have cancer, or have had a cancer treated in the past (e.g.,treated with surgical resection, chemotherapy, radiation therapy). Forexample, Zscane4⁺ ES or iPS cells can be used in patients who have had atumor removed, wherein specific cells differentiated from Zscan4⁺ ES oriPS cells are used to reconstruct the removed tissues/organs. Inaddition, as genome instability is often associated with cancers, agentsthat can induce or enhance Zscan4 expression (e.g., expression of anexogenous Zscan4 nucleic acid molecule in the cancer cell) or activateZscan4 pathways can be administered to prevent cancer cells frombecoming more aggressive due to genome instability.

Exemplary cancers that can benefit from the Zscan4⁺ ES or iPS cellsprovided herein include but are not limited to cancers of the heart(e.g., sarcoma (angiosarcoma, fibrosarcoma, rhabdomyosarcoma,liposarcoma), myxoma, rhabdomyoma, fibroma, lipoma and teratoma), lung(e.g., bronchogenic carcinoma (squamous cell, undifferentiated smallcell, undifferentiated large cell, adenocarcinoma), alveolar(bronchiolar) carcinoma, bronchial adenoma, sarcoma, lymphoma,chondromatous hamartoma, mesothelioma); gastrointestinal tract (e.g.,esophagus (squamous cell carcinoma, adenocarcinoma, leiomyosarcoma,lymphoma), stomach (carcinoma, lymphoma, leiomyosarcoma), pancreas(ductal adenocarcinoma, insulinoma, glucagonoma, gastrinoma, carcinoidtumors, vipoma), small bowel (adenocarcinoma, lymphoma, carcinoidtumors, Karposi's sarcoma, leiomyoma, hemangioma, lipoma, neurofibroma,fibroma), large bowel (adenocarcinoma, tubular adenoma, villous adenoma,hamartoma, leiomyoma), genitourinary tract (e.g., kidney(adenocarcinoma, Wilm's tumor, nephroblastoma, lymphoma, leukemia),bladder and urethra (squamous cell carcinoma, transitional cellcarcinoma, adenocarcinoma), prostate (adenocarcinoma, sarcoma), testis(seminoma, teratoma, embryonal carcinoma, teratocarcinoma,choriocarcinoma, sarcoma, interstitial cell carcinoma, fibroma,fibroadenoma, adenomatoid tumors, lipoma), liver (e.g., hepatoma(hepatocellular carcinoma), cholangiocarcinoma, hepatoblastom,angiosarcoma, hepatocellular adenoma, hemangioma), bone (e.g.,osteogenic sarcoma (osteosarcoma), fibrosarcoma, malignant fibroushistiocytoma, chondrosarcoma, Ewing's sarcoma, malignant lymphoma(reticulum cell sarcoma), multiple myeloma, malignant giant cell tumor,chordoma, osteochronfroma (osteocartilaginous exostoses), benignchondroma, chondroblastoma, chondromyxofibroma, osteoid osteoma andgiant cell tumors), nervous system (e.g., skull (osteoma, hemangioma,granuloma, xanthoma, osteitis deformans), meninges (meningioma,meningiosarcoma, gliomatosis), brain (astrocytoma, medulloblastoma,glioma, ependymoma, germinoma >pinealoma!, glioblastoma multiforme,oligodendroglioma, schwannoma, retinoblastoma, congenital tumors),spinal cord (neurofibroma, meningioma, glioma, sarcoma)), gynecologicalcancers (e.g., uterus (endometrial carcinoma), cervix (cervicalcarcinoma, pre-tumor cervical dysplasia), ovaries (ovarian carcinoma,serous cystadenocarcinoma, mucinous cystadenocarcinoma, endometrioidtumors, celioblastoma, clear cell carcinoma, unclassified carcinoma,granulosa-thecal cell tumors, Sertoli-Leydig cell tumors, dysgerminoma,malignant teratoma), vulva (squamous cell carcinoma, intraepithelialcarcinoma, adenocarcinoma, fibrosarcoma, melanoma), vagina (clear cellcarcinoma, squamous cell carcinoma, botryoid sarcoma, embryonalrhabdomyosarcoma, fallopian tubes (carcinoma)), hematologic cancers(e.g., blood (myeloid leukemia (acute and chronic), acute lymphoblasticleukemia, chronic lymphocytic leukemia, myeloproliferative diseases,multiple myeloma, myelodysplastic syndrome), Hodgkin's disease,non-Hodgkin's lymphoma (malignant lymphoma)), skin (e.g., malignantmelanoma, basal cell carcinoma, squamous cell carcinoma, Karposi'ssarcoma, moles, dysplastic nevi, lipoma, angioma, dermatofibroma,keloids, psoriasis), and adrenal glands (e.g., neuroblastoma).

In one example, a patient with an autoimmune disease is selected fortreatment. Autoimmune diseases can result from an overactive immuneresponse of the body against substances and tissues normally present inthe body. In some examples, the autoimmune disease is be restricted tocertain organs (e.g., in thyroiditis) or can involve a particular tissuein different places (e.g., Goodpasture's disease which may affect thebasement membrane in both the lung and the kidney). Patients treatedwith the Zscan4⁺ ES cells disclosed herein may have an autoimmunedisease. Exemplary autoimmune diseases that can benefit from the Zscan4⁺ES cells provided herein include but are not limited to, rheumatoidarthritis, juvenile oligoarthritis, collagen-induced arthritis,adjuvant-induced arthritis, Sjogren's syndrome, multiple sclerosis,experimental autoimmune encephalomyelitis, inflammatory bowel disease(for example, Crohn's disease, ulcerative colitis), autoimmune gastricatrophy, pemphigus vulgaris, psoriasis, vitiligo, type 1 diabetes,non-obese diabetes, myasthenia gravis, Grave's disease, Hashimoto'sthyroiditis, sclerosing cholangitis, sclerosing sialadenitis, systemiclupus erythematosis, autoimmune thrombocytopenia purpura, Goodpasture'ssyndrome, Addison's disease, systemic sclerosis, polymyositis,dermatomyositis, autoimmune hemolytic anemia, and pernicious anemia.

In some examples, the subject selected is one who has suffered aneurologic injury or suffers from a neurodegenerative disorder.Neurologic injuries can result from a trauma to the nervous system (suchas to the brain or spinal cord or particular neurons), which adverselyaffects the movement and/or memory of the injured patient. Such traumasmay be caused by an infectious agent (e.g., a bacterium or virus), atoxin, an injury due to a fall or other type of accident, or geneticdisorder, or for other unknown reasons. Patients treated with the EScells disclosed herein may have suffered a neurologic injury, such as abrain or spinal cord injury resulting from an accident, such as anautomobile or diving accident, or from a stroke.

A neurodegenerative disease is a condition in which cells of the brainand spinal cord are lost. Patients treated with the ES cells disclosedherein may have a neurodegenerative disease. Exemplary neurodegenerativediseases that can be treated with the Zscan4+ ES cells provided hereininclude but are not limited to: adrenoleukodystrophy (ALD), alcoholism,Alexander's disease, Alper's disease, Alzheimer's disease, amyotrophiclateral sclerosis (Lou Gehrig's Disease), ataxia telangiectasia, Battendisease (also known as Spielmeyer-Vogt-Sjogren-Batten disease), bovinespongiform encephalopathy (BSE), Canavan disease, cerebral palsy,Cockayne syndrome, Corticobasal degeneration, Creutzfeldt-Jakob disease,familial fatal insomnia, frontotemporal lobar degeneration, Huntington'sdisease, HIV-associated dementia, Kennedy's disease, Krabbe's disease,Lewy body dementia, neuroborreliosis, Machado-Joseph disease(Spinocerebellar ataxia type 3), Multiple System Atrophy, multiplesclerosis, narcolepsy, Niemann Pick disease, Parkinson's disease,Pelizaeus-Merzbacher Disease, Pick's disease, primary lateral sclerosis,prion diseases, progressive supranuclear palsy, Refsum's disease,Sandhoff disease, Schilder's disease, subacute combined degeneration ofspinal cord secondary to Pernicious Anaemia,Spielmeyer-Vogt-Sjogren-Batten disease (also known as Batten disease),spinocerebellar ataxia, spinal muscular atrophy,Steele-Richardson-Olszewski disease, Tabes dorsalis, toxicencephalopathy.

Zscan4⁺ ES or iPS cells can be obtained or generated using the methodsdescribed herein. Methods of administering ES or iPS cells to mammaliansubjects are known in the art. For example, Zscan4⁺ ES cells or iPS canbe administered to a subject in need of such therapy via injection, suchas subcutaneous, intramuscular, intradermal, intraperitoneal,intravenous or intra-arterial administration. In some examples, Zscan4⁺ES or iPS cells are administered directly to the area in need oftreatment, such as to a cancerous organ or tissue, or to the brain orspinal cord. In some examples, Zscan4⁺ ES or iPS cells are administeredalone, in the presence of a pharmaceutically acceptable carrier (such asencapsulated in a suitable polymer) or in the presence of othertherapeutic agents. In some embodiments, a subject is administered atleast 20,000 Zscan4⁺ ES cells, such as at least 50,000, at least100,000, at least 500,000, at least 1,000,000, or at least 2,000,000Zscan4⁺ ES cells.

In one example, Zscan4⁺ ES or iPS cells are encapsulated into asemipermeable polymer membrane and the polymer membrane transplantedinto a tissue site of a host subject. Such method may achieve local,long-term chronic delivery of a therapeutic substance with thecapability of regulating release of the substance. See U.S. Pat. No.5,573,528 for description of encapsulation of compounds and cells. Inone embodiment, Zscan4⁺ ES cells are encapsulated within a polymermembrane. The encapsulated polymer membrane is then transplanted into atissue site of a host subject. In one example, the tissue site iscentral nervous system, such as brain or spinal cord.

The semipermeable polymer membrane can be synthetic or natural. Examplesof polymer that can be used include polyethersulfone (PES),polyacrylonitrile-co-vinyl chloride (P[AN/VC], poly(lactic acid),poly(lactic-co-glycolic acid), methylcellulose, hyaluronic acid,collagen, and the like. Delivery of encapsulated Zscan4⁺ ES cells withina polymer membrane can avoid host rejection and immune response tocells, and problems associated with rejection and inflammation. Inaddition, cells contained within the polymer membrane are shielded bythe wall of the polymer (i.e., the walls of the individual fibers,fibrils, films, sprays, droplets, particles, etc.) from immunesurveillance while still maintaining cell viability and allowingtransport of molecules, nutrients and metabolic products through thepolymer walls. The grafting of polymer-encapsulated cells has beendeveloped by Aebischer et al., 1991, Transplant, 111:269-275, and hasbeen successfully used with both non-human primates and humans(Aebischer et al., 1994, Transplant, 58:1275-1277). See also U.S. Pat.No. 6,110,902.

In one example, Zscan4⁺ ES or iPS cells are encapsulated by firstembedding them into a matrix of either collagen, agarose or PVA(polyvinylalcohol). Subsequently, the embedded cells are injected intohollow fibers made of polypropylene of a 60:40 copolymer ofpolyacrylnitrile:polyvinylchloride. The fibers are cut into pieces andend-sealed for implantation. In one example, the encapsulated cells haveabout 20,000 to about 2,000,000 Zscan4⁺ ES cells.

In some examples, the Zscan4⁺ ES or iPS cells are of exogenous origin.By the term “exogenous” is meant cells obtained from sources other thanthe subject in which they are implanted for treatment. Exogenous cellscan be from other organisms of the same species (such as human-derivedcells for use in a human patient). Exogenous cells can also be fromheterologous sources, i.e., from a species distinct from the subject tobe therapeutically treated (such as mouse cells for use in a human).Zscan4⁺ ES or iPS cells can also be taken from an isogenic source, i.e.,from the subject who is to receive the cells. After harvesting the cellsfrom the subject, the cells can be genetically modified (e.g., a nucleicacid encoding Zscan4 is introduced) or selected/enriched for Zscan4⁺ ESor iPS cells, then reimplanted back to the subject. Since the cells areisogeneic, no immune response is expected.

In one aspect, the Zscan4⁺ ES or iPS cells are immortalized. For exampleand not by way of limitation, cells can be conditionally immortalized(such that the cells grow well in tissue culture at reducedtemperatures, yet discontinue division once implanted into a patient andmaintained at 37° C.) or constitutively immortalized (e.g., transfectionwith constructs expressing large T antigen, or immortalization byEpstein Barr virus) by methods well known in the art. Another method ofdelivering Zscan4⁺ ES or iPS cells into a host subject is to directlytransplant the cells into the target area of a tissue site. Oncetransplanted, these cells survive, migrate and integrate seamlessly intothe host tissue. In one example, the Zscan4⁺ ES or iPS cells aredirectly transplanted into the nervous system of the host subject, suchas a developing nervous system or a nervous system that has suffered atrauma or in a subject having a neurological disorder. When transplantedinto a developing nervous system, the Zscan4⁺ ES cells will participatein processes of normal development and will respond to the host'sdevelopmental cues. The transplanted neural precursor cells will migratealong established migratory pathways, will spread widely intodisseminated areas of the nervous system and will differentiate in atemporally and regionally appropriate manner into progeny from both theneuronal and glial lineages in concert with the host developmentalprogram. The transplanted Zscan4⁺ ES or iPS cell is capable ofnon-disruptive intermingling with the host neural precursor cells aswell as differentiated cells. The transplanted cells can replacespecific deficient neuronal or glial cell populations, restore defectivefunctions and can express foreign genes in a wide distribution.

The Zscan4⁺ ES or iPS cells can also be transplanted into a developednervous system. The transplanted neural precursor cells can form astable graft, migrate within the host nervous system, intermingle andinteract with the host neural progenitors and differentiated cells. Theycan replace specific deficient neuronal or glial cell populations,restore deficient functions and activate regenerative and healingprocesses in the host's nervous system. In one example, the stable graftis a graft established in the central nervous system or the peripheralnervous system.

Similar methods can be used to directly transplant Zscan4⁺ ES or iPScells into any region in need of ES cell therapy. Such cells may beundifferentiated or differentiated into the desired cell type in vitro(then administered to a subject in need thereof). For example, whereorgan regeneration is desired, for example for replacement of organs ortissues removed to treat cancer or lost for other reasons (e.g., teeth,hair, cells of the ear or eyes, skin or muscle). In one example, Zscan4⁺ES or iPS cells are directly transplanted into the heart, for example toregenerate cardiac tissue or cells lost to myocardial infarction. In oneexample, Zscan4⁺ ES or iPS cells are directly transplanted into thepancreas, for example to regenerate cells in a subject with diabetes. Inone example, Zscan4⁺ ES or iPS cells are directly transplanted into thebone or administered systemically, for example to regenerate bone marrowcells in a subject having cancer.

The therapeutic dose and regimen most appropriate for patient treatmentwill vary with diseases or conditions to be treated, and according tothe patient's weight and other parameters. An effective dosage andtreatment protocol can be determined by conventional means, startingwith a low dose in laboratory animals and then increasing the dosagewhile monitoring the effects, and systematically varying the dosageregimen. Numerous factors can be taken into consideration by a clinicianwhen determining an optimal dosage for a given subject. Factors includethe size of the patient, the age of the patient, the general conditionof the patient, the particular disease being treated, the severity ofthe disease, the presence of other drugs in the patient, and the like.The trial dosages would be chosen after consideration of the results ofanimal studies and the clinical literature.

Accordingly, Zscan4⁺ ES or iPS cells are administered to subjects so asto reduce or ameliorate symptoms associated with a particular disorder.Therapeutic endpoints for the treatment of cancer can include areduction in the size or volume of a tumor, reduction in angiogenesis tothe tumor, or reduction in metastasis of the tumor. If the tumor hasbeen removed, another therapeutic endpoint can be regeneration of thetissue or organ removed. Effectiveness of cancer treatment can bemeasured using methods in the art, for example imaging of the tumor ordetecting tumor markers or other indicators of the presence of thecancer. Therapeutic endpoints for the treatment of autoimmune diseasescan include a reduction in the autoimmune response. Effectiveness ofautoimmune disease treatment can be measured using methods in the art,for example measuring of autoimmune antibodies, wherein a reduction insuch antibodies in the treated subject indicates that the therapy issuccessful. Therapeutic endpoints for the treatment of neurodegenerativedisorders can include a reduction in neurodegenerative-related deficits,e.g., an increase in motor, memory or behavioral deficits. Effectivenessof treating neurodegenerative disorders can be measured using methods inthe art, for example by measuring cognitive impairment, wherein areduction in such impairment in the treated subject indicates that thetherapy is successful. Therapeutic endpoints for the treatment ofneurologic injuries can include a reduction in injury-related deficits,e.g., an increase in motor, memory or behavioral deficits. Effectivenessof treating neurologic injuries can be measured using methods in theart, for example by measuring mobility and flexibility, wherein anincrease in such in the treated subject indicates that the therapy issuccessful. Treatment does not require 100% effectiveness. A reductionin the disease (or symptoms thereof) of at least about 10%, about 15%,about 25%, about 40%, about 50%, or greater, for example relative to theabsence of treatment with Zscan4⁺ ES or iPS cells, is consideredeffective.

In some examples, Zscan4⁺ ES or iPS cells are administered at a dosefrom about 1×10⁴ cells to about 1×10⁷ cells in a mouse or other smallmammal, or a dose from about 1×10⁴ cells to about 1×10¹⁰ cells in ahuman or other large mammal. In one specific, non-limiting example, atherapeutically effective amount is about 1×10⁶ cells. Other therapeuticagents (for example, chemical compounds, small molecules, or peptides)can be administered in a therapeutically effective dose in combinationwith the Zscan4⁺ ES cells (for example shortly before or after, orsimultaneously) in order to achieve a desired effect in a subject beingtreated. An effective amount of Zscan4⁺ ES cells may be administered ina single dose, or in several doses, for example daily, during a courseof treatment. However, one skilled in the art will appreciate that theeffective amount of Zscan4⁺ ES cells will be dependent on the agentapplied, the subject being treated, the severity and type of theaffliction, and the manner of administration of the agent.

IX. Use of Zscan4 Polypeptides and Polynucleotides

It is disclosed herein that expression of Zscan4 increases genomestability, protects cells against DNA damage and enhances DNA repair.Thus, provided herein is a method of treating a subject with cancer byadministering to the subject a Zscan4 polypeptide or polynucleotide. Insome embodiments, the method further includes selecting a patient inneed of such therapy, such as a subject that has been diagnosed withcancer.

In some embodiments of the methods disclosed herein, the subject isadministered a Zscan4 polynucleotide. In some examples, the subject isadministered a vector comprising a Zscan4 polynucleotide. Methods ofgenerating and using Zscan4-expressing vectors are described in othersections of the application. In some cases, the Zscan polynucleotide (orvector comprising the Zscan4 polynucleotide) is administered directly totumor cells to tumor tissue, such as by injection.

In other embodiments, subject is administered a Zscan4 polypeptide. Insome examples, the Zscan4 polypeptide is encapsulated by a nanoparticleto aid in delivery of the Zscan4 polypeptide to tumor cells. Suitablenanoparticles for use with the disclosed methods are known in the artand are described below.

Nanoparticles are submicron (less than about 1000 nm) sized drugdelivery vehicles that can carry encapsulated drugs such as syntheticsmall molecules, proteins, peptides and nucleic acid basedbiotherapeutics for either rapid or controlled release. A variety ofmolecules (e.g., proteins, peptides and nucleic acid molecules) can beefficiently encapsulated in nanoparticles using processes well known inthe art.

The nanoparticles for use with the compositions and methods describedherein can be any type of biocompatible nanoparticle, such asbiodegradable nanoparticles, such as polymeric nanoparticles, including,but not limited to polyamide, polycarbonate, polyalkene, polyvinylethers, and cellulose ether nanoparticles. In some embodiments, thenanoparticles are made of biocompatible and biodegradable materials. Insome embodiments, the nanoparticles include, but are not limited tonanoparticles comprising poly(lactic acid) or poly(glycolic acid), orboth poly(lactic acid) and poly(glycolic acid). In particularembodiments, the nanoparticles are poly(D,L-lactic-co-glycolic acid)(PLGA) nanoparticles.

PLGA is a FDA-approved biomaterial that has been used as resorbablesutures and biodegradable implants. PLGA nanoparticles have also beenused in drug delivery systems for a variety of drugs via numerous routesof administration including, but not limited to, subcutaneous,intravenous, ocular, oral and intramuscular. PLGA degrades into itsmonomer constituents, lactic and glycolic acid, which are naturalbyproducts of metabolism, making the material highly biocompatible. Inaddition, PLGA is commercially available as a clinical-grade materialfor synthesis of nanoparticles.

Other biodegradable polymeric materials are contemplated for use withthe compositions and methods described herein, such as poly(lactic acid)(PLA) and polyglycolide (PGA). Additional useful nanoparticles includebiodegradable poly(alkylcyanoacrylate) nanoparticles (Vauthier et al.,Adv. Drug Del. Rev. 55: 519-48, 2003). Oral adsorption also may beenhanced using poly(lactide-glycolide) nanoparticles coated withchitosan, which is a mucoadhesive cationic polymer. The manufacture ofsuch nanoparticles is described, for example, by Takeuchi et al. (Adv.Drug Del. Rev. 47: 39-54, 2001).

Among the biodegradable polymers currently being used for humanapplications, PLA, PGA, and PLGA are known to be generally safe becausethey undergo in vivo hydrolysis to harmless lactic acid and glycolicacid. These polymers have been used in making sutures when post-surgicalremoval is not required, and in formulating encapsulated leuprolideacetate, which has been approved by the FDA for human use (Langer andMose, Science 249:1527, 1990); Gilding and Reed, Polymer 20:1459, 1979;Morris, et al., Vaccine 12:5, 1994). The degradation rates of thesepolymers vary with the glycolide/lactide ratio and molecular weightthereof. Therefore, the release of the encapsulated molecule (such as aprotein or peptide) can be sustained over several months by adjustingthe molecular weight and glycolide/lactide ratio of the polymer, as wellas the particle size and coating thickness of the capsule formulation(Holland, et al., J. Control. Rel. 4:155, 1986).

In some embodiments, the nanoparticles for use with the compositions andmethods described herein range in size from about 50 nm to about 1000 nmin diameter. In some cases, the nanoparticles are less than about 600nm. In some embodiments, the nanoparticles are about 100 to about 600 nmin diameter. In some embodiments, the nanoparticles are about 200 toabout 500 nm in diameter. In some embodiments, the nanoparticles areabout 300 to about 450 nm in diameter. One skilled in the art wouldreadily recognize that the size of the nanoparticle may vary dependingupon the method of preparation, clinical application, and imagingsubstance used.

Various types of biodegradable and biocompatible nanoparticles, methodsof making such nanoparticles, including PLGA nanoparticles, and methodsof encapsulating a variety of synthetic compounds, proteins and nucleicacids, has been well described in the art (see, for example, U.S.Publication No. 2007/0148074; U.S. Publication No. 20070092575; U.S.Patent Publication No. 2006/0246139; U.S. Pat. No. 5,753,234; U.S. Pat.No. 7,081,489; and PCT Publication No. WO/2006/052285).

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the disclosure to the particular features or embodimentsdescribed.

EXAMPLES Example 1 Experimental Procedures

ES Cell Culture

MC1 ES cells, derived from 12956/SvEvTac (Olson et al., Cancer Res 63,6602-6606, 2003), were purchased from the Transgenic Core Laboratory ofthe Johns Hopkins University School of Medicine, Baltimore, Md., USA.R26R3ES cells (Soriano et al., Nat. Genet. 21:70-71, 1999) were used asthe parental line to generate pZscan4-CreERT2 cells for lineage trackingexperiments. In general, all ES cell lines were cultured for 2 passageson gelatin-coated feeder-free plates before further experiments and weresubsequently maintained in gelatin-coated 6-well plates in complete ESmedium: DMEM (Gibco), 15% FBS (Atlanta Biologicals); U/ml leukemiainhibitory factor (LIF) (ESGRO, Chemicon); 1 mM sodium pyruvate; 0.1 mMnon-essential amino acids; 2 mM GlutaMAX™; 0.1 mM beta-mercaptoethanol;and penicillin/streptomycin (50 U/50 μg/ml). For all cell lines, mediumwas changed daily and cells were routinely passaged every 2 to 3 days.

pZscan4-Emerald Vector Construction

A genomic region spanning 3563 bp upstream to the Zscan4c startmethionine was selected as the putative-Zscan4 promoter. This DNA regionwas amplified from BAC RP23-63I1 with high fidelity TITANIUM™ Taq(Clontech), using a forward primer (GGCAACCTTACTACTTCTATC; SEQ ID NO: 1)modified with a MluI cutting sequence at the 5′ end, and a reverseprimer (AGCATCAACCACTTTGGTAC; SEQ ID NO: 2). Subsequently, the PCRproduct was cloned into the pcDNA6.2/C-EmGFP TOPO™ vector (Invitrogen).Sequence-verified plasmid DNA was linearized by MluI digestion (to alsoremove the cytomegalovirus promoter) prior to cloning of the Zscan4promoter region. The nucleotide sequence of the resulting vector is setforth as SEQ ID NO: 38.

Generation of pZscan4-Emerald ES and pZscan4-CreERT2 Cells

MC1 ES cells (for pZscan4-Emerald transfection) and R26R6ES cells (forpZscan4-Cre ERT2 transfection) were grown on gelatin in 6-well plates,5×10⁵ cells in suspension were transfected with 1 μg of linearizedpZscan4-Emerald vector or pZscan4-CreERT2 vector using Effectene™(QIAGEN) according to manufacturer's protocol, and plated ingelatin-coated 100 mm dishes. Cells were selected with 5 μg/mlblasticidin, colonies were picked on the 8th day, expanded and frozenfor further analysis.

Sorting pZscan4-Emerald ES Cells

Cells were fed at least 2 hours before harvesting by Accutase™(Chemicon) and resuspended in Iscove's Modified Dulbecco's Medium (IMDM)containing 25 mM HEPES buffer (Chemicon) with 1% FBS and 1000 U/ml LIF.Cells were FACS-sorted according to Emerald intensity. The same gatingwas used for all experiments. The cells were sorted into IMDM with 35%serum, 1 mM sodium pyruvate, 2 mM GlutaMAX™, 100 μM β-mercaptoethanol,100 U/ml Penicillin, 100 μg/ml streptomycin, 0.1 mM non-essential aminoacids and 1000 U/ml LIF. For microarray experiments, RNA was collectedimmediately after sorting by TRIZOL™ (Invitrogen) following themanufacturer's protocol.

Double-Fluorescence RNA in Situ Hybridization

Digoxigenin (DIG)- and Biotin (BIO)-labeled RNA probes were transcribedfrom the PCR product templates using RNA Labeling Mix (Roche).Ethanol-precipitated probes were resuspended in water and quantified byRNA 6000 Nano Assay on a 2100 Bioanalyzer™ (Agilent Technologies). Cells(10⁵ cells/well) were seeded in glass chamber slides, cultured for threedays, fixed with paraformaldehyde (PFA), and permeabilized with 0.5%Triton X-100. Cells were washed and incubated with 1 μg/ml DIG and BIOprobes for 12 hours at 60° C. in hybridization solution. Probes weredetected by mouse anti-DIG antibody and by sheep anti-BIO antibody, andvisualized by fluorophore-conjugated secondary antibodies. Nuclei werestained with DAPI (blue).

pZscan4-CreERT2 a Zscan4 Lineage-Tracing Vector Construction

A pCre-ERT2 ORF was subcloned into the EcoRI site of pBluescriptllSK(+)plasmid (Feil et al., Biochem. Biophys. Res. Commun. 237:752-7, 1997).Subsequently, the PCR fragment of the Zscan4c promoter was subclonedbetween EcoRV and EcoRI, located at the 5′-end of the Cre-ERT2 ORF, byblunt-end ligation. A SalI-NotI fragment containing the Zscan4cpromoter-Cre-ERT2 was then subcloned into the HindIII-PmeI fragment ofpEF6/V5-His-TOPO™ by blunt-end ligation.

Tracking Zscan4 Expressing Cell-Fate using pZscan4-Cre ERT2 System

pZscan4-Cre ERT2 ES cells were grown in standard ES medium containing100 nM tamoxifen. Biological triplicates at passages 1, 2, 3, 4 and 9were stained for beta-galactosidase using a commercial kit (Chemicon)according to the manufacturer's protocol. In addition, cells maintainedfor a longer period in the presence of tamoxifen were harvested atpassages 1, 2, 3 and 4, stained for beta-galactosidase with DetectaGene™Green CMFDG LacZ Gene Expression kit (Invitrogen) and analyzed usingGuava EasyCyte Mini flow cytometry system with the CytoSoft4.1 software(Guava Technologies).

Zscan4 Lineage Tracing Via Differentiation of Embryoid Bodies

For lineage tracing in embryoid bodies (EB) formation assay (Doetschmanet al., J. Embryol. Exp. Morphol. 87:27-45, 1985), pZscan4-Cre ERT2 EScells were grown on gelatin for 3 days in complete medium containing 100nM tamoxifen, harvested and 4×10⁶ ES were plated on 100 mmbacteriological Ultra-Low Culture Dish (Corning) in LIF-free mediumwithout tamoxifen to form floating EB. On the 7th day, floating EB werecollected and plated in gelatin coated 6-well plates in LIF-free mediumwithout tamoxifen to allow attachment. On the 11^(th) day, beating areaswere scored and subsequently cells were fixed in 4% PFA for LacZstaining and immunohistochemistry.

Quantitative Reverse Transcriptase Polymerase Chain Reaction (qRT-PCR)

RNA was isolated from cells by TRIZOL™ (Invitrogen) in biologicaltriplicate. Total RNA (1 μg) was reverse transcribed by Superscript IIIfollowing the manufacturer's protocol, using 100 ng of oligo dT primer(Promega) per reaction. For qPCR, SYBR green master mix (AppliedBiosystems) was used following the manufacturer's protocol, with 96-welloptical plates, a 25 μl total reaction volume and 10 ng cDNA per well.Plates were run on 7900HT or 7500 system (Applied Biosystems). Foldinduction was calculated by the ΔΔCt method (Livak and Schmittgen,Method. Methods 25:402-8, 2001) using H2A as normalizer, unlessotherwise noted.

RNA Isolation, cDNA Preparation and qPCR Analysis in MousePreimplantation Embryos

Four to six week-old B6D2F1 female mice were super-ovulated with 5 IU ofpregnant mare's serum gonadotropin (PMSG; Sigma) and 5 IU of humanchorionic gonadotropin (hCG; Sigma). Eggs or embryos for qRT-PCRexperiments were collected after 20, 23, 30, 43, 55, 66, 80 and 102hours post-hCG injection for MII, 1-cell, early and late-2 cell, 4-cell,8-cell, morula and blastocyst embryos, respectively. Three sets of 10synchronized eggs or embryos were stored in liquid nitrogen andmechanically ruptured by a freeze/thaw step for the cDNA preparationtemplate. Oligo-dT primers and Super Script III Reverse Transcriptase(Invitrogen) were used according to the manufacturer's instructions.Analysis was performed on the ABI 7500 Fast Real Time PCR system(Applied Biosystems). A list of qPCR primer sequences are shown below inTable 1. Data was normalized by Chuk with the ΔΔCt method (Falco et al.,Reprod. Biomed. Online 13:394-403, 2006; Livak and Schmittgen, Method.Methods 25:402-8, 2001).

TABLE 1 qPCR Primer Sequences Primer Sequence SEQ ID NO:AF067063 (forward) tcagagggggccacaagtgttc  3 AF067063 (reverse)cagaccaacccttgccaagctt  4 BC061212 (forward) ccatgcaaggtgtccactttctcac 5 BC061212 (reverse) ggggtccctctccatcactcacta  6 Eif1a (forward)tgctcgtccggctgtgacgg  7 Eif1a (reverse) gctctcagaagccaggactctgca  8Gm428 (forward) tgtacgagatcttgggccaccc  9 Gm428 (reverse)cacggtaacccaccagcttcctat 10 Tho4 (forward) atggacacttggaggcagccag 11Tho4 (reverse) gacgcccagctggatgcttatc 12 Arginase II (forward)agaccacagcctggcaatag 13 Arginase II (reverse) aaggtaccacaactgccagg 14Tcstv3_v1 (forward) tctccagctgttgtggaataagttcaac 15 Tcstv3_v1 (reverse)cttcttggctttatccatggatccctgaaggtaaatc 16 Zscan4 (forward)cctccctgggcttcttggcat 17 Zscan4 (reverse) agctgccaaccagaaagacactgt 18Chuk (forward) caggaccgtgttctcaaggagctg 19 Chuk (reverse)gctctggtcctcatttgcttcacg 20 Primers were designed with the Vector NTIsoftware (Invitrogen, Carlsbad, CA) and tested using ovarian cDNAmixtures with Syber Green PCR Master Mix (Applied Biosystem, FosterCity, CA) as previously described (Falco et al., Reprod. Biomed. Online13: 394-403, 2006).Generation of ROSA/Empty Knock-in Parental-Cell Clones

MC1 ES cells were grown on feeders and harvested on day 3. Cells (10⁷)were electroporated with 26 μg of linearized pMWRosaTcH vector usingGene Pulser Xcell™ electroporation system (BioRad). Subsequently, cellswere plated on gelatin-coated feeder-free plates. Cells were selectedfor 10 days with 100 μg/ml of hygromycin. Seventeen resistant colonieswere picked and knock-in was confirmed by Southern blot using randomprimed ³²P-labeled external and internal probes (Masui et al., NucleicAcids Res. 33:e43, 2005).

Construction of ROSA/Tet-Off Zscan4-Flag Targeting Vector and Generationof Tet-Zscan4c Cells and Tet-Empty Cells

The backbone pZhcSfi plasmid (Masui et al., Nucleic Acids Res. 33:e43,2005) was modified, and the Zeocin-resistance gene was replaced by thepuromycin-resistance gene. PGKpA was replaced by SV40pA derived frompIRESpuro3 (Clontech) and inserted into the multiple cloning site.Zscan4c ORF fragments were amplified by PCR and sub-cloned into themodified pZhcSfi. A 6×His-FLAG epitope tag sequence was inserted intothe 5′ end of LoxPV to flank Zscan4c-fused C-terminal epitope tag. TheORF of Zscan4c was verified by sequencing. MC1 Rosa26 knock-in parentalES cells were co-transfected with the modified pZhcSfi carrying Zscan4cORF and pCAGGS-Cre plasmid using Effectene™ (QIAGEN) according tomanufacturer's instruction and selected by puromycin in the presence ofdoxycycline (0.2 μg/ml). Clones were isolated and thereafter namedtet-Zscan4c cells. Modified pZhcSfi without the ORF was used toestablish tet-Empty control cells.

RNA in Situ Hybridization

In situ hybridization was carried out as previously described (Carter etal., Gene Expr. Patterns 8:181-198, 2008). Briefly, tet-Zscan4c cells intriplicate, grown in the presence or absence of doxycycline for 3 days,were fixed in 4% PFA at 4° C. overnight. After digestion with proteinaseK, cells were hybridized with 1 μg/ml digoxigenin-labeled riboprobe at62° C. overnight. Cells were then washed, blocked, incubated withalkaline phosphatase-conjugated anti-digoxigenin antibody, and incubatedwith NBT/BCIP detection buffer for 30 minutes.

Zscan4 Knockdown Vector Construction

For Zscan4 knockdown experiments, four different 19-mer sequences fromthe Zscan4 gene were designed as shRNA for Zscan4 with a 19-mer senseoligo, a hairpin loop and an anti-sense of the same sequence. The mostefficient sequence was:

(SEQ ID NO: 21) Primer forward:CATAACCTGAAAAAACAGAAGCCTGGCATTCCCTAAGCTTAGGGAATGCCAGGCTTCTGCGCGTCCTTTCCACAAGATATATA, (SEQ ID NO: 22) Primer reverse:CATAACCTGAAAAAACAGAAGCCTGGCATTCCCTTTCGAAAGGGAATGCCAGGCTTCTGCGCGTCCTTTCCACAAGATATATA.

shRNAs were amplified with GeneSilencer U6-GFP PCR kit (Gelantis)according to the manufacturer protocol. The shRNAs were intended totarget a common sequence in the 3′-UTR of Zscan4c and Zscan4d paralogsto allow rescue by exogenous Zscan4 expression. Initially, tet-Zscan4ccells were transiently transfected by Effectene™ (QIAGEN) according tomanufacturer's protocol; GFP was used as a reported gene fortransfection efficiency. Cells were selected with hygromycin and cloneswere isolated to establish Zscan4 knockdown and rescue cells. To achieverescue cells were incubated with complete ES medium without Dox for 3days.

RNA was isolated; cDNA was made as described and tested by qPCR tomeasure Zscan4 expression.

Microarray Analysis

DNA microarray analysis of pZscan4-Emerald cells was carried out asdescribed (Aiba et al., DNA Res. 16:73-80, 2009). In brief, universalmouse reference RNA (Stratagene) was labeled with Cy5-dye, mixed withCy3-labeld samples, and used for hybridization on the NIA Mouse 44KMicroarray v2.2 (Carter et al., Genome Biol. 6:R61, 2005) (manufacturedby Agilent Technologies #014117). The intensity of each gene feature wasextracted from scanned microarray images using Feature Extraction9.5.1.1 software (Agilent Technologies). Microarray data analyses werecarried out by using an application developed to perform ANOVA and otheranalyses (NIA Array Analysis software; see lgsun.grc.nia.nih gov/ANOVA/)(Sharov et al., Bioinformatics 21:2548-9, 2005). All the DNA microarraydata have been deposited in the NCBI Gene Expression Omnibus (GEO,http://www.ncbi.nlm nih gov/geo/) and are accessible through GEO Seriesaccession number (GSE#15604) and the NIA Array Analysis software website(lgsun.grc.nia.nih gov/ANOVA/) (Sharov et al., Bioinformatics 21:2548-9,2005).

Generation of Zscan4 Antibodies

Polyclonal Rabbit anti-Zscan4 antibodies (Genescript), were generatedaccrording to the manufacturer's protocol, against the N-terminalepitope of Zscan4: LQTNNLEFTPTDSSC (SEQ ID NO: 39).

Telomere Quantitative-Fluorescence In-Situ Hybridization (Q-FISH)

All cells were maintained in complete ES medium containing doxycycline.For Zscan4 induction, doxycycline was removed from the medium for atotal of 3 days. Medium was replaced every day. On the 3^(rd) day,medium was supplemented with colcemid 0.1 μg/ml (Invitrogen) andincubated for 4 hours to arrest the cells in metaphase. Hypotonic 0.075M KCl buffer was added to samples and then cells were fixed in coldmethanol:acetic acid at a ratio of 3:1. Metaphase spreads were prepared.Telomere FISH was performed by Telomere peptide nucleic acid (PNA) FISHKit/Cy3 (DakoCytomation) according to the manufacturer's protocol.Chromosomes were stained with 0.5 μg ml DAPI. Chromosomes and telomereswere digitally imaged on a Zeiss microscope with Cy3-DAPI filter sets.For quantitative measurement of telomere length, telomere size andfluorescence intensity was evaluated by TFL-TELO software (Poon et al.,Cytometry 36:267-278, 1999).

Telomere Chromosome-Orientation FISH(CO-FISH)

Chromatid orientation (CO)—FISH analysis was performed as previouslydescribed (Bailey et al., Mutagenesis 11:139-44, 1996; Goodwin andMeyne, Cytogenet. Cell. Genet. 63:126-7, 1993) with several minormodifications. Briefly, ES cells were grown in either Dox+ or Dox−conditions for 3 days induction. Medium was changed every day. On thethird day, 5′-bromo-2′-deoxyuridine (BrdU) was added for 12 hours toallow BrdU incorporation for one cell cycle. Colcemid 0.1 μg/ml wasadded for the final 4 hours. Metaphase spreads were prepared. Slideswere stained with 0.5 μg/ml of Hoechst 33258 (Sigma), washed in 2×saline-sodium citrate (SSC) buffer for 20 minutes at room temperature,mounted with Mcllvaine's buffer (at pH 8.0), and exposed to 365-nmultraviolet (UV) light (Stratelinker 1800 UV irradiator) for 30 minutes.The BrdU-substituted DNA was digested with 3 units/μl of Exonuclease III(Promega) for 10 minutes at room temperature. The leading strandtelomeres were revealed by 3′-Cy3-conjugated (TTAGGG)₇ (SEQ ID NO: 23),without a denaturation step and incubated overnight at 37° C.Chromosomes were counterstained with 1 μg/ml DAPI (Vector Laboratories).

Telomere Measurement by Quantitative Real-Time PCR

Genomic DNA was extracted from 10⁶ cells and quantified by Nanodrop.Average telomere length ratio was measured from total genomic DNA usinga real-time PCR assay, as previously described (Callicott and Womack,Comp. Med. 56:17-22, 2006). PCR reactions were performed on the Prism7500 Sequence Detection System (Applied Biosystems), using telomericprimers, control single-copy gene Rp1p0 and PCR settings as previouslydescribed (Id.). A standard curve was made for the reference gene byserial dilutions of known amounts of DNA from 100 ng to 3.125 ng. Thetelomere signal was normalized to RplpO to generate a T/S ratioindicative of relative telomere length.

Telomerase Activity Measurement

Cells were cultured in triplicate on gelatin-coated dishes for 3 days incomplete ES medium in the presence (Dox+) or absence of doxycycline(Dox−). Cell lysates were prepared from 10⁶ cells per replica.Telomerase activity was measured by telomeric repeat amplificationprotocol (TRAP) assay using a TRAPEZE™ Telomerase Detection Kit(Millipore) according to the manufacturer's instruction.

Karyotype Analysis

ES cell cultures were treated with 0.1 μg/ml colcemid (Invitrogen) for 2hours to induce metaphase arrest. Metaphase chromosome spreads wereprepared and stained with 0.3% Giemsa reagent for 20 minutes, andchromosomes were counted for n≧40 metaphases per sample. Results forknockdown and control cells were also verified by DAPI-staining intelomere FISH analyzed cells.

Sister Chromatid Exchange (SCE) Assay

SCE assay was performed as previously described (Perry and Wolff, Nature251:156-8, 1974). Briefly, mouse ES cells were maintained in complete ESmedium containing doxycycline. For Zscan4 induction, doxycycline wasremoved from the medium for total of 3 days. Medium with BrdU was addedfor the last 24 hours, allowing the cells to complete two cell cycles.Medium was supplemented with 0.1 μg/ml colcemid for the last 4 hours toarrest the cells in metaphase. Metaphase spreads were prepared and takenfor SCE analysis. SCE were counted in n>50 metaphases per sample and theexperiment was repeated for 3-4 independent experiments per sample(total of n>150 metaphases).

Immunofluorescence Staining Analysis

Cells were plated in 24-well plates on sterilized coverslips. Replicateswere maintained in doxycycline medium (Dox+) while for three replicatesdoxycycline was removed (Dox−) for 3 days in order to induce Zscan4over-expression. Medium was changed every day. Cells were either fixedin 4% PFA for 10 minutes at room temperature or taken for metaphasespreads as described above. Cells in PFA were permeabilized with 0.25%NP-40 for 10 minutes. Cells were blocked for 10 minutes at roomtemperature in 1% BSA, 10% FBS, and 0.2% saponin and incubated overnightat 4° C., with the primary antibodies: anti-FLAG antibody diluted1:1000, anti-ZSCAN4 1:400, anti-SPO11 1:200, anti DMC1 1:200, anti-TRF11:500, anti-TRF2 1:400 in blocking solution. As negative controls, cellsstained without primary antibody were used, as well as the Dox+ cellsstained with anti-FLAG antibody. The bound antibody was visualized witha fluorescent Alexa546 secondary antibody (Invitrogen) under a Zeiss510-confocal microscope. Nuclei were visualized with DAPI (Roche)staining for 5 minutes at room temperature.

Example 2 Zscan4 is Expressed in 5% of ES Cells in UndifferentiatedConditions

RNA in situ hybridization of Zscan4 showed a highly heterogeneousstaining pattern in MC1 mouse ES cell colonies (FIG. 1A) (Carter et al.,Gene Expr. Patterns 8:181-198, 2008; Falco et al., Dev. Biol.307:539-550, 2007). As a first step to characterize Zscan4-expressing EScells, a reporter plasmid designed to express the green fluorescenceprotein (GFP)-Emerald under Zscan4c promoter (FIG. 1B) was transfectedinto MC1 ES cells and a stable transformant, pZscan4-Emerald cells, wasisolated. As expected, Emerald expression was observed in a small numberof the ES cells in culture, recapitulating previous observations by RNAin situ hybridization (FIG. 1C). FACS analysis indicated thatapproximately 5% of the ES cells were Emerald-positive (FIG. 1D),although the number slightly varied (2-7%) even when the same cultureconditions were used. Quantitative real-time reverse-transcriptionpolymerase chain reaction (qRT-PCR) analysis of FACS-sorted cellsdemonstrated about 1.000-fold enrichment of Zscan4 mRNA in Emerald(+)(Em(+)) cells relative to Em(−) cells. Furthermore, when the cells weresorted into subgroups according to respective Emerald intensity, adirect correlation between Emerald-fluorescence intensity and Zscan4mRNA levels was observed (FIG. 1E), thus establishing that Em(+) cellsare also Zscan4(+) cells.

Example 3 Zscan4 Expression Marks a Transient and Reversible State inUndifferentiated ES Cells

To investigate whether Zscan4 expression marks distinctive cell typeswithin ES cell colonies, pZscan4-Emerald cells were FACS-sorted intoEm(+) cells and Em(−) cells, and subsequently replated separately in astandard ES cell culture medium. Both Em(+) and Em(−) cells were able toestablish viable, undifferentiated ES cell colonies. However, by 24hours in culture 54% of the Em(+) cells became Em(−), whereas 3.3% ofEm(−) cells became Em(+) as shown by FACS analysis (FIGS. 1F-1H).Furthermore, live-cell imaging on pZscan4-Emerald cells in a time-lapseexperiment documented the transition of cells between the two states,and confirmed that both Em(+) and Em(−) cells can replicate properly andestablish new colonies with a heterogeneous Emerald expression pattern.Therefore, Zscan4 is expressed transiently in ES cells, and thetransition between Zscan4(+) state and Zscan4(−) state is reversible.The transient Zscan4(+) state of ES cells is referred to herein as“ES-star (ES*) state.”

Example 4 ES* State is Associated with Up-Regulation of Early-EmbryonicMarkers

ES* state was further characterized by DNA microarray analysis ofFACS-sorted Em(+) and Em(−) cells. The Em(+) cells showed a very similargene expression profile to the Em(−) cells with only 550 differentiallyexpressed genes (FIG. 1I). Pluripotency-related markers remainedunchanged in Em(+) cells compared to Em(−) cells, but Testv1 and Tcstv3(two cell-specific transcript variant 1 and 3) genes (GenBank accessionAF067057.1; Zhang et al., Nucleic Acids Res. 34:4780-90, 2006) werefound among the top 20 most highly up-regulated genes (FIG. 1J).

RNA in situ hybridization revealed “Zscan4-like” expression for eightother genes in the list (Tcstyl/3, Eif1a, Pif1, AF067063, EG668777,LOC332923, BC061212, and EG627488) (FIG. 2A). Furthermore,double-labeling fluorescence RNA in situ hybridization confirmedco-expression of these genes with Zscan4 (FIG. 2B). As Zscan4 is a2-cell embryo marker, the expression profile of six genes from the top20 genes most highly upregulated in Em(+) cells were examined by qRT-PCRin preimplantation embryos. All six genes examined showed a highexpression peak in 2-cell embryos; 3 genes showed the highest peak atthe late 2-cell stage as Zscan4, whereas 3 others showed the highestexpression at the early 2-cell stage (FIG. 2C). Taken together, theseresults indicate that some of the early-stage embryo programs arereactivated in the ES* state.

Example 5 Most ES Cells in Culture Go through the ES* State

To trace the fate of ES* cells, a plasmid carrying CreERT2 (Feil et al.,Biochem. Biophys. Res. Commun. 237:752-7, 1997) driven by the Zscan4cpromoter was transfected into a ROSA26-knockin ES cell line carrying afloxed neomycin cassette in a LacZ open reading frame (ORF) (Soriano,Nat. Genet. 21:70-71, 1999) and a stable cell line, pZscan4-CreERT2 EScells, isolated (FIG. 3A). In this system, Cre-recombinase is expressedin cells in the ES* state, translocates from the cytoplasm into thenucleus only in the presence of tamoxifen, and excises a neomycincassette from LacZ-ORF, leading to a constitutive LacZ expression (FIG.3A). Cells in the ES* state are thus heritably labeled with LacZ.

As expected, cells positive for Zscan4 RNA by fluorescence in situhybridization were co-stained with nuclear-localized Cre-recombinaseprotein as demonstrated by immunostaining (FIG. 3B). To identify thetotal population of cells marked by Zscan4 expression, pZscan4-CreERT2ES cells were maintained continuously in undifferentiated conditions inthe presence of tamoxifen for 9 passages (27 days). Immunostaininganalysis of the cells demonstrated that Pou5f1 (Oct4 or Oct 3/4) wasco-stained with LacZ, indicating that they were still undifferentiated(FIG. 3C). Visualization of LacZ activity by X-gal staining showed thatthe number of cells marked with LacZ steadily increased with passagesand the majority of ES cell colonies became LacZ-positive by passage 9(FIGS. 3D and 3F). These observations were further confirmed by FACSanalysis following CMFDG staining (a green fluorescence LacZ substrate)(FIGS. 3E and 3F). Taken together with the observation frompZscan4-Emerald ES cells, cell fate tracing experiments confirmed thatat a given time only 5% of ES cells are positive for Zscan4 expression,but after 9 passages most ES cells in culture experience Zscan4expression (i.e., ES* state).

Example 6 Cells in ES* State Maintain Pluripotency in Vitro and in Vivo

To determine if Zscan4 expression leads to a certain cell lineagecommitment in ES cells, pZscan4-CreERT2 cells were exposed to a pulse oftamoxifen, followed by embryoid body (EB) formation and attachment assay(Doetschman et al., J. Embryol. Exp. Morphol. 87:27-45, 2007). In the EBdifferentiation, cells derived from the ES* state were able tocontribute to a variety of cell types including lineages from all threeembryonic germ layers, as judged by cell morphology as well asimmunostaining for specific lineage markers (FIGS. 3G and 3H).

To test the pluripotency of cells in ES* state in vivo, FACS-sortedEm(+) cells were microinjected into 16 mouse blastocysts and four pupswere obtained, two of which were chimeric based on coat color. Inanother experiment, 8 out of 9 embryos (E10.5) produced were chimericbased on genotyping by PCR analysis (FIG. 3I). Therefore, the datademonstrate that cells in ES* state retain their pluripotency in vitroand in vivo, as they can contribute to all cell lineages after in vitrodifferentiation by EB formation, and have the ability to form chimera invivo. Taken together, these results indicate that physiological Zscan4expression in cell culture is transient and reversible inundifferentiated ES cells, and does not affect the pluripotency of thecells.

Example 7 Zscan4c Knockdown Leads to a Cell Culture Crisis of ES Cellsby 8 Passages

To directly test whether the intermittent expression of Zscan4 isessential for ES cells, Zscan4-knockdown and -rescue cells weregenerated. First, tet-Zscan4c ES cells were generated by integrating atetracycline (tet)-repressible ORF of Zscan4c into the ROSA26 locus ofthe mouse genome using the Cre-LoxP system (Masui et al., Nucleic AcidsRes. 33:e4, 2005). In the tet-Zscan4c ES cells, a Venus reporter gene,linked to Zscan4c RNA by an IRES, allowed monitoring gene induction.Subsequently, tet-Zscan4c ES cells were transfected with a Zscan4-shRNAvector targeting a common 3′-untranslated region of both Zscan4c andZscan4d, and ES cell clones with normal morphology were isolated. Thesystem was designed to knockdown the expression of both Zscan4c andZscan4d in the presence of doxycycline (Dox+) and to provide a rescue byexpressing the exogenous copy of Zscan4c-ORF by doxycycline removal(Dox−). qRT-PCR analysis confirmed the downregulation of Zscan4cexpression by 90±2.4% as well as Zscan4d by 70±7.0% (FIG. 4A). Culturingthe cells in the Dox− condition for 3 days induced Zscan4c-ORFexpression by 5.9±0.22 fold, whereas the control shRNA did not affectZscan4 gene expression in the same parental cells. Zscan4c inductionalso resulted in upregulation of Zscan4d, a paralog expressedpredominantly in the 2-cell embryo, suggesting a positive feedbackbetween the two paralogs (FIG. 4A).

As expected from the transient and intermittent expression of Zscan4,Zscan4-knockdown did not affect the ES cells immediately and showedtypical ES cell morphology upon clonal isolation (FIG. 4B). However,with passages, the population doubling time of the Zscan4-knockdowncells became longer than control cells and flat non-dividing cells beganto accumulate in culture (FIGS. 4C and 4D). During passage 8(approximately 31 cell doublings after transfection), most of the cellsdied abruptly 1-2 days after plating, leaving very few surviving smallcolonies (FIGS. 4C and 4D). It was possible to recover the survivingcolonies by passaging the cells every 3 days without splitting for twoweeks. However, the surviving cells had an abnormally long doublingtime. This phenotype was reproducible in multiple independentexperiments.

Example 8 Decreased Cell Proliferation in Zscan4-Knockdown Cells

To investigate the event leading to cell culture crisis during passage8, cell proliferation and apoptosis assays were performed at passage 7.The presence or absence of Dox alone did not affect the proliferationrate of the cells, as all the control cells, including tet-Empty cells,tet-Zscan4c cells, and shRNA control cells in Dox+ and Dox− conditions,showed normal proliferation curves (FIG. 4E). In contrast, a significantreduction in proliferation was observed in Zscan4-knockdown cell lines 1and 2 in Dox+ conditions (FIG. 4E). A rescue experiment by induction ofthe exogenous copy of Zscan4c in Dox− condition was able to recover cellproliferation (FIG. 4E).

Apoptosis and viability tests were further performed on Zscan4-knockdowncell lines at passage 7 and control cells daily for three days (FIG.4F). Although the level of apoptosis in Zscan4-knockdown cells washigher than those (up to 2%) in all control cells (i.e., tet-Zscan4cparental cells, tet-Empty cells in Dox+ and Dox− conditions as well asshRNA control cells), it reached only up to 14%, suggesting that thesignificant reduction of cell numbers by Zscan4 knockdown is causedmostly by the reduction of cell proliferation, but not by the apoptosis.In addition, this moderate increase of apoptosis rate was not rescued bythe induction of Zscan4c-ORF in the Dox− condition, suggesting that theincrease in apoptosis rate was not a direct effect of Zscan4 knockdown.

Example 9 Karyotype Deterioration and Genomic Instability inZscan4-Knockdown Cells

Zscan4-knockdown (cell line 2) and control cells were analyzed forkaryotype at passage 3, the earliest passage expanded following cloneisolation (FIG. 4G). In contrast to control cells (Dox+) with 82.5%normal karyotype, Zscan4-knockdown cells (Dox+) presented only 65%euploid or normal karyotype with multiple abnormalities includingvarious types of chromosome fusions and fragmentations. Although this isstill considered acceptable for cultured ES cells, it was apparent thatthe rescue cells (Dox−) showed better karyotype (75% normal). Controlcells and parental cells kept under similar conditions did not exhibitsimilar abnormalities (FIG. 4G). Similar abnormalities were observed forthe Zscan4-knockdown cell line 1 (Table 2), suggesting that genomicinstability was not due to a specific clonal defect, but caused byZscan4 knockdown. When cells were karyotyped at passage 10 after cellculture crisis, only 35% of the Zscan4-knockdown cells showed normalkaryotype. Remarkably, rescue cells were significantly better,presenting 60% normal karyotype.

TABLE 2 Additional Data for Karyotype Analysis Karyotype AnalysisControl Cells Zscan4-knockdown Zscan4-knockdown (Passage 10) cell line 1(Passage 3) cell line 1 (Passage 10) tet- tet- tet- tet- Zscan4 Zscan4Empty Empty Zscan4 Zscan4 Knock- Zscan4 Knock- Zscan4 (Dox+) (Dox−)(Dox+) (Dox−) down Rescue down Rescue No. of 58 57 55 61 40 40 40 40metaphases examined No. of 7 7 5 3 16  6 28 16 metaphases withabnormalities Mean 39.93 39.22 39.89 39.97   39.75  39.8   39.55   39.65chromosome no./ metaphase % of cells with 87.9% 87.7% 90.9% 95.1%  60% 75%  30%  60% normal karyotype in culture Similar to the data for cellline 2 shown in FIG. 4G, at passage 3 the Zscan4-knockdown cellspresented multiple karyotype abnormalities, such as fusions andfragmentations, whereas Zscan4-rescue cells had improved karyotype. Thekaryotype at passage 10 after cell crisis further deteriorated, whereasthat of Zscan4-rescue cells was 60% normal. Parental tet-Zscan4 cells,used as control, showed a slight improvement of karyotype after Zscan4induction (in Dox− condition), whereas tet-Empty control cells hadsimilar results in both Dox+ and Dox− conditions.

Example 10 Abnormally Short Telomeres in Zscan4-Knockdown ES Cells

The compromised karyotype in Zscan4-knockdown cells prompted examinationof their telomeres by telomere fluorescence in situ hybridization (FISH)(FIG. 5A) and telomere qPCR (FIG. 5B). For qPCR, telomere length ratioswere compared to a single copy gene as previously described (Callicottand Womack, Comp. Med. 56:17-22, 2006; Cawthon, Nucleic Acids Res. 30:e47, 2002). Cells were collected in two different passages before andafter cell crisis.

Remarkably, both telomere FISH and telomere qPCR showed a significanttelomere shortening in both Zscan4-knockdown cell line 1 and 2. qPCRanalysis showed that average telomere length decreased by 30±5%(mean±S.E.M.) in cell line 1 and 45±0.1% in cell line 2 at passage 6(FIG. 5B). In contrast, the telomeres of shRNA control cells under thesame conditions, as well as tet-Empty cells and the parental tet-Zscan4ccells in non-induced Dox+ conditions, remained intact with passages.qPCR data indicated that telomere shortening was rescued by Zscan4cover-expression (FIG. 5B), suggesting that knockdown of Zscan4 causestelomere shortening.

Telomere qPCR data were further validated by quantitative fluorescencein situ hybridization (Q-FISH) analysis (Poon et al., Cytometry36:267-278, 1999). Telomeres were visualized by Cy3-conjugated probes(FIG. 5A) and the signal intensity was measured by the TFL-Telosoftware. Telomere length distribution diagram demonstrated that overalltelomere lengths in the Zscan4-knockdown cells at passage 6 (FIG. 5C)were shorter than those in the control cells (FIG. 5E).

Surprisingly, telomeres were elongated in Zscan4-rescue cells byoverexpressing Zscan4c; on average they were 1.7-fold longer than thosein the normal cells and 2.4-fold longer than those in the knockdowncells (FIG. 5C). The distribution diagram for Zscan4-rescue cells alsoindicates that the average increase of telomere length was not due toabnormally long telomere, but due to an overall shift to the longer, yetnormal spectrum of telomere length (FIG. 5C).

Example 11 Transient Expression of Zscan4 Extends Telomere Length in ESCells

The effects of Zscan4 over-expression on telomeres was also examined inthe parental tet-Zscan4c cells before and after gene induction (FIGS. 5Dand 5E). Indeed, telomere Q-FISH results showed a significant increase(1.8-fold) in the average-telomere length from 39.4±0.13 kb(mean±S.E.M.) in the non-induced tet-Zscan4c cells to 65.9±0.2 kb inZscan4 overexpressing cells (FIG. 5E). Consistent with the results ofZscan4 rescue shown in FIG. 5C, the telomere length distribution diagramindicated a shift to a longer but normal spectrum of telomere length(FIG. 5E).

To verify Q-FISH results, telomeres were also measured by telomere qPCR.In accordance to Q-FISH results, the relative telomere length intet-Zscan4c cells in Dox− condition was greater (2.47±0.38-fold) thanthat in non-induced Dox+ condition (FIG. 5F). As expected, this effectwas not caused by the Dox itself, as control tet-Empty cells did notshow a significant difference between Dox+ and Dox− conditions.Furthermore, telomere qPCR analysis was able to correlate telomereextension to the endogenous ES* state using FACS-sorted pZscan4-Emeraldcells (FIG. 5F). The relative telomere length of Em(+) cells was2.44±0.27-fold longer than control tet-Empty cells, whereas that ofEm(−) cells was 0.89±0.15-fold shorter (FIG. 5F). Taken together, thetelomere Q-FISH and qPCR data indicate that ES* state (i.e., Zscan4cexpression) is associated with extended telomere length.

To test the possibility that telomere lengthening by Zscan4 is mediatedby an increase in telomerase activity, Telomeric Repeat AmplificationProtocol (TRAP) assay was conducted. TRAP assay demonstrated only a mildeffect (30% higher) on telomerase activity in the induced Dox−tet-Zscan4c cells compared to non-induced Dox+ cells (FIG. 6A). However,tet-Empty cells also gave a similar mild response to Dox removal (FIG.6A). Therefore, the slight increase of telomerase activity may berelated to Dox removal, but not directly caused by Zscan4 induction.Furthermore, it is unlikely that the slight increase in telomeraseactivity could explain the rapid telomere elongation associated withZscan4 over-expression.

Example 12 ZSCAN4 is Co-Localized with Meiosis-Specific HomologousRecombination Proteins on Telomeres

The predicted structure of ZSCAN4 indicates the presence of SCAN domainas well as a DNA-binding domain indicating a role in recruitment ofother proteins to chromatin. Indeed, immunostaining analysis showednuclear localization of ZSCAN4C-FLAG after a 3-day induction by Doxremoval (FIG. 6B). To determine whether ZSCAN4 protein is active onchromatin during the M phase, metaphase chromosome spreads ofZscan4-overexpressing cells before and after gene induction was assayedby immunostaining analysis (FIG. 6C). ZSCAN4C-FLAG was localized to themetaphase chromosomes; in particular, some of the chromosomes had moreintense staining at both ends of chromosomes, suggesting more specificlocalization to telomeres of these chromosomes.

To test whether ZSCAN4 protein functions in telomere recombination, aco-localization study was performed for ZSCAN4 with telomere markers.Immunostaining analysis by confocal microscopy demonstrated ZSCAN4 fociwere co-labeled on telomeres with TRF1 (Chong et al., Science270:1663-7, 1995) and TRF2 (Opresko et al., J. Biol. Chem. 277:41110-9,2002) (FIG. 8A). Additionally, microarray analysis indicated thatmeiosis-specific homologous recombination genes were induced by Zscan4cover-expression in ES cells. Analyses by qRT-PCR validated the resultsfor the enzyme Spo11 (Keeney et al., Cell 88:375-84, 1997; Mahadevaiahet al., Nat. Genet. 27:271-6, 2001), which facilitates the double strandDNA breaks (DSBs) during meiotic recombination, the recombinase Dmc1(Reinholdt et al., Chromosoma 114:127-34, 2005), required for DSBsrepair, and the cohesion Smc1 (Revenkova et al., Nat. Cell Biol.6:555-62, 2004) (FIG. 8B). Immunostaining showed SPO11 was co-localizedwith ZSCAN4 foci (FIG. 8C, upper panel), indicating SPO11 plays a rolein induction of DSBs in telomeres to initiate T-SCEs. During meioticrecombination, DSBs are enclosed in—H2AX/ZSCAN4 foci forming afterZscan4c over-expression (FIG. 8C, lower panel). The RecA homolog DMC1was further co-localized with TRF1 in Em+ cells (FIG. 8D, lower panel);moreover, ZSCAN4 foci were confirmed to be co-localized with TRF1 in Em+cells (FIG. 8D, upper panel). Taken together, these results indicatethat Zscan4 is involved in the induction and recruitment of themeiosis-specific homologous recombination machinery to telomeres.

Example 13 Zscan4 Over-Expression Promotes Telomere Recombination

A recent discovery has established that preimplantation embryos have theability to activate rapid telomere extension, within 1 cell cycle,through telomere sister chromatid exchange (T-SCE) (Liu et al., Nat.Cell Biol. 9:1436-41, 2007). As Zscan4 is a common marker for ES cellsand 2-cell embryos, it was investigated whether the alternativelengthening of telomeres (ALT) is activated in ES* state by carrying outtelomere chromosome-orientation FISH(CO-FISH) (Bailey et al.,Mutagenesis 11:139-144, 1996) (FIG. 6D). The frequency of telomererecombination in the tet-Empty cells (control) was very low and showedno significant difference between Dox+ and Dox− conditions: 0.19±0.13(Mean±S.E.M.). T-SCE events per nucleus were seen in Dox+ condition and0.24±0.14 events per nucleus, in the Dox− condition (FIG. 6E). A similarfrequency was observed for the non-induced tet-Zscan4c cells in the Dox+condition: 0.26±0.15 T-SCE events/nucleus were found. In contrast,Zscan4c induction for 3 days resulted in >10-fold increase in T-SCEevents: 76% of the nuclei showed 2.97±0.21 T-SCE events/nucleus (FIG.6E). Therefore, the data clearly indicated that transient expression ofZscan4 promoted the telomere recombination.

Example 14 Increased Incidence of Sister Chromatid Exchange inZscan4-Knockdown Cells

To further examine the genome instability of Zscan4-knockdown cells, asister chromatid exchange (SCE) assay was performed. A significantlyhigher frequency of SCE events was found in Zscan4-knockdown cells (FIG.7A). Tet-Empty cells as well as parental tet-Zscan4c cells were used ascontrols.

Consistent with previous reports on ES cells (Dronkert et al., Mol.Cell. Biol. 20:3147-56, 2000; Tateishi et al., Mol. Cell. Biol.23:474-81, 2003), the basal SCE was relatively low in all control cells:tet-Empty cells had no significant difference by Dox removal with only5.25±2.55 SCE events per SCE-positive nucleus (i.e., 13±1.4% of allcells analyzed) in Dox+ (FIGS. 7B and 7C) and 6.25±2.65 SCE events perSCE-positive nucleus (i.e., 12.7±3% of all cells analyzed) in the Dox−condition (FIGS. 7B and 7C). The basal SCE rate for the parentaltet-Zscan4c cells in the Dox+ condition was slightly lower and only 8±2%of the nuclei had 4.75±1.26 SCE/positive nucleus. In contrast, 29±0.7%of Zscan4-knockdown cells had 11.27±4.2 SCE/positive nucleus (FIGS. 7Band 7C). When Zscan4 expression was rescued by the Dox− condition for 3days, the number of spontaneous SCE was dramatically reduced. Only 13%of the metaphase spreads were SCE-positive (FIG. 7B) with a small number(2.86±2.1) of SCE per SCE-positive nucleus (FIG. 7C), which wascomparable to the control cells.

Surprisingly, Zscan4c over-expression in parental tet-Zscan4c cellsreduced SCE events dramatically and only 2±1.15% of the cells had just2.25±1.5 SCE events/positive nucleus, which was 4-fold lower than thatin the Dox+ condition and 6-fold lower than in the tet-Empty controls(FIGS. 7B and 7C). The data indicate that the expression of Zscan4 candecrease the incidence of SCE in ES cells, whereas knockdown increasesgenomic instability and SCE rate (FIG. 7D).

Taken together, these data provide multiple lines of evidence thatZscan4 is indispensable for long-term genomic stability, for examplethrough mechanisms including the SCE rate as well as telomereregulation.

Example 15 Retinoic Acids Induce Transient Expression of Zscan4

This example describes the finding that retinoids (including vitamin A,13-cis-retinoic acid, 9-cis-retinoic acid, and all-trans-retinoic acid)can transiently increase Zscane cells in mouse ES cell culture.

Materials

A 20 mM stock solution of all-trans retinoic acid (atRA) in ethanol wasused at a final concentration of 1 μM, 2 μM, or 50 nM. 9-cis RA and13-cis RA were used at a final concentration of 1 μM final. Vitamin A(retinol) was used at a final concentration of 5 μM.

Results

Zscan4 Expression is Induced Transiently by All-Trans Retinoic Acid(atRA)

Mouse ES cells (referred to as pZscan4-Emerald7 (MC1-ZE7) cells) inwhich a plasmid carrying a green fluorescent protein (Emerald: Em)regulated under the promoter of Zscan4c is integrated in the genome wereused in these studies. The inventors' earlier work showed that theinduction of Zscan4 expression can be monitored by the induction ofEmerald fluorescence. Cells were first passaged to gelatin-coated platesin the presence of leukemia inhibitory factor (LIF+) and absence of atRA(atRA−). The next day (Day 0), the culture medium of each well waschanged to four different conditions: LIF+atRA−, LIF+atRA+, LIF− atRA−,and LIF− atRA+. The final concentration of atRA was 2 M. The cells weremaintained in the same culture medium for 8 days with medium changesevery day, but without passaging. Cells were harvested every day and thenumber of Emerald GFP⁺ cells was measured by flow cytometry (Guava).

The fraction of Em⁺ cells (i.e., Zscan4⁺ cells) in the culture wasdramatically induced by atRA and peaked on the second day of atRAtreatment (FIG. 9). However, this induction of Zscan4⁺ cells wastransient and declined to the basal level by the fourth day of atRAtreatment. LIF is a well-established factor that can maintain ES cellsin an undifferentiated state and is a part of the standard ES cellculture medium. The transient induction of Zscan4⁺ cells by atRA wasobserved in both LIF+ and LIF− conditions.

Zscan4 Expression is Induced Transiently by Other Retinoids

Although atRA is most commonly used for mammalian cell culture systems,there are other retinoids that sometimes have similar activities on thecells. To test other retinoids for Zscan4-inducibility, similartime-course kinetic assays were carried out using atRA (50 nM finalconcentration), 9-cis RA (50 nM final concentration), 13-cis RA (50 nMfinal concentration), or vitamin A (5 M final concentration). Thefraction of Zscan4⁺ cells was transiently increased and peaked on day 2by each retinoid (FIG. 10A). However, within 7 days of treatment, thesecondary increase of Zscane cells was observed for vitamin A and 13-cisRA, but not for atRA and 9-cis RA. This could be partially explained bydifferential effects of these retinoids on cell proliferation; vitamin Adid not affect cell proliferation, 13-cis RA showed moderate suppressionof cell proliferation, and both atRA and 9-cis RA induced nearlycomplete suppression of cell proliferation (FIG. 10B). Zscan4⁺ cells mayhave growth advantages over Zscan4⁻ cells in the presence of vitamin Aand 13-cis RA.

These findings demonstrate that retinoids (including vitamin A,13-cis-RA, 9-cis-RA, and atRA) can transiently increase Zscan4⁺ cells inmouse ES cell culture.

Example 16 Oxidative Stress Induces Zscan4 Expression

This example describes the finding that expression of Zscan4 isincreased in ES cells exposed to oxidative stress.

MC1-ZE7 cells were cultured in standard ES cell medium (LIF+). Hydrogenperoxide (H₂O₂) was added to the medium at a final concentration of 100μM, 300 μM, or 1000 μM. The cells were cultured for two days and thepercentage of Em⁺ (i.e., Zscan4⁺ cells) was determined by the Guava flowcytometry (Guava). The results demonstrate that oxidative stress (asinduced by H₂O₂) increases the number of Zscan4⁺ cells (FIG. 11).

Example 17 Zscan4 Protects ES Cells Against DNA-Damaging Agents

This example describes the finding that overexpression of Zscan4 in EScells enhances survival of the cells following exposure to DNA-damagingagents, including mitomycin C (MMC) and cisplatin.

Control cells (carrying the tet-Empty plasmid) and Zscan4-expressingcells (carrying the tet-Zscan4 plasmid) were used in this study. Thecells were cultured in standard ES medium (LIF+). Cells were passagedinto two groups: (1) in the absence of doxycycline (Dox−), or (2) in thepresence of doxycycline (Dox+) at a final concentration of 0.2 μg/ml.The Dox+ and Dox− media were changed every day. On the fourth day, thecells were cultured for 6 hours in the presence of MMC at a finalconcentration ranging from 0 to 600 ng/ml. The MMC was then removed fromthe culture by replacing the media. The cells were incubated for 3 moredays in the Dox+ medium, and the medium was changed every day. Cellswere harvested and the number of live cells was counted.

The results show that in control cells, the higher the dose of MMC, thelower the cell survival rate (FIG. 12A). However, there are nosignificant difference between Dox+ and Dox− conditions for controlcells (in which Zscan4 expression is not induced). In contrast, therewere significant difference between Dox+ and Dox− conditions in theZscan4-expressing ES cells (FIG. 12B). These cells exhibited increasedsurvival against the MMC treatment. These results indicate thatZscan4-overexpression protects ES cells from genotoxic assaults byDNA-damaging agents, such as MMC. Similar results were obtained withcisplatin.

In view of the many possible embodiments to which the principles of thedisclosure may be applied, it should be recognized that the illustratedembodiments are only examples of the disclosure and should not be takenas limiting the scope of the invention. Rather, the scope of theinvention is defined by the following claims. We therefore claim as ourinvention all that comes within the scope and spirit of these claims.

The invention claimed is:
 1. A method for increasing genome stability ofan isolated mouse embryonic stem (ES) cell; or increasing telomerelength in an isolated mouse ES cell; or both, comprising transfecting anisolated nucleic acid molecule encoding mouse Zscan4c into the ES cellunder conditions sufficient to allow for expression of mouse Zscan4c inthe ES cell.
 2. The method of claim 1, wherein the isolated nucleic acidmolecule comprises a vector.
 3. The method of claim 2, wherein thevector encodes mouse Zscan4c operably linked to a promoter.
 4. Themethod of claim 1, wherein the nucleotide sequence of mouse Zscan4c isat least 95% identical to the nucleotide sequence of SEQ ID NO:
 28. 5.The method of claim 1, wherein the nucleotide sequence of mouse Zscan4ccomprises the nucleotide sequence of SEQ ID NO:
 28. 6. The method ofclaim 1, wherein the nucleotide sequence of mouse Zscan4c consists ofthe nucleotide sequence of SEQ ID NO:
 28. 7. The method of claim 2,wherein the vector is a viral vector.
 8. The method of claim 2, whereinthe vector is a plasmid vector.
 9. The method of claim 3, wherein thepromoter is a constitutive promoter.
 10. The method of claim 3, whereinthe promoter is an inducible promoter.
 11. The method of claim 1,wherein expression of mouse Zscan4c is transient.
 12. The method ofclaim 1, wherein the isolated nucleic acid molecule encoding mouseZscan4c is integrated into the genome of the ES cell.